Amphipols, Nanodiscs, and Fluorinated Surfactants: Three ...Fluorinated Surfactants: Three...

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Amphipols, Nanodiscs, andFluorinated Surfactants:Three NonconventionalApproaches to StudyingMembrane Proteins inAqueous SolutionsJean-Luc PopotLaboratoire de Physico-Chimie Moleculaire des Proteines Membranaires, Unite Mixte deRecherche 7099, Centre National de la Recherche Scientifique and Universite Paris-7 DenisDiderot, Institut de Biologie Physico-Chimique, F-75005 Paris, France;e-mail: [email protected]

Annu. Rev. Biochem. 2010. 79:737–75

First published online as a Review in Advance onMarch 18, 2010

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev.biochem.052208.114057

Copyright c© 2010 by Annual Reviews.All rights reserved

0066-4154/10/0707-0737$20.00

Key Words

nanolipoprotein particles, nanoscale apolipoprotein-bound bilayers,reconstituted high-density lipoprotein particles, hemifluorinatedsurfactants

AbstractMembrane proteins (MPs) are usually handled in aqueous solutions asprotein/detergent complexes. Detergents, however, tend to be inacti-vating. This situation has prompted the design of alternative surfactantsthat can be substituted for detergents once target proteins have beenextracted from biological membranes and that keep them soluble inaqueous buffers while stabilizing them. The present review focuses onthree such systems: Amphipols (APols) are amphipathic polymers thatadsorb onto the hydrophobic transmembrane surface of MPs; nanodiscs(NDs) are small patches of lipid bilayer whose rim is stabilized by am-phipathic proteins; fluorinated surfactants (FSs) resemble detergents butinterfere less than detergents do with stabilizing protein/protein andprotein/lipid interactions. The structure and properties of each of thesethree systems are described, as well as those of the complexes they formwith MPs. Their respective usefulness, constraints, and prospects forfunctional and structural studies of MPs are discussed.

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Contents

1. INTRODUCTION: STRANGERIN A STRANGE LAND . . . . . . . . . . 738

2. KEEPING MEMBRANEPROTEINS WATER SOLUBLEIN THE ABSENCE OFDETERGENTS . . . . . . . . . . . . . . . . . . 741

3. PRINCIPLE AND MOLECULARORGANIZATION OFNONCONVENTIONALSURFACTANTS . . . . . . . . . . . . . . . . . . 7423.1. Nanodiscs . . . . . . . . . . . . . . . . . . . . . 7423.2. Fluorinated Surfactants . . . . . . . . 7433.3. Amphipathic Polymers

(Amphipols) . . . . . . . . . . . . . . . . . . . . 7454. TRANSFERRING MEMBRANE

PROTEINS TONONCONVENTIONALSURFACTANTS . . . . . . . . . . . . . . . . . . 747

5. STRUCTURE OF MEMBRANEPROTEIN ANDNONCONVENTIONALSURFACTANT COMPLEXES. . . . 7485.1. Membrane Protein/A8-35

Complexes . . . . . . . . . . . . . . . . . . . . . 7485.2. Membrane Protein/Nanodisc

Complexes . . . . . . . . . . . . . . . . . . . . . 7515.3. Membrane Protein/

(Hemi)Fluorinated SurfactantComplexes . . . . . . . . . . . . . . . . . . . . . 751

6. FUNCTIONALITY ANDSTABILITY OF MEMBRANEPROTEINS INNONCONVENTIONALSURFACTANTS . . . . . . . . . . . . . . . . . . 752

6.1. Membrane Protein/AmphipolComplexes . . . . . . . . . . . . . . . . . . . . . 752

6.2. MembraneProtein/HemifluorinatedSurfactant Complexes . . . . . . . . . . . 753

6.3. Membrane Protein/NanodiscComplexes . . . . . . . . . . . . . . . . . . . . . 755

7. APPLICATIONS . . . . . . . . . . . . . . . . . . 7557.1. Constraints for Optical

Spectroscopy . . . . . . . . . . . . . . . . . . . 7557.2. Solution Studies of Membrane

Protein Mass, Shape, andInteractions. . . . . . . . . . . . . . . . . . . . . 756

7.3. Functional Studies . . . . . . . . . . . . . 7567.4. Proteomics: Isoelectrofocusing,

Two-Dimensional Gels,and Mass Spectrometry . . . . . . . . . 757

7.5. Nuclear Magnetic ResonanceStudies . . . . . . . . . . . . . . . . . . . . . . . . . 758

7.6. Crystallization . . . . . . . . . . . . . . . . . 7607.7. Single-Particle Electron

Microscopy and AtomicForce Microscopy . . . . . . . . . . . . . . . 761

7.8. Delivering Membrane Proteinsto Preformed Lipid Bilayers . . . . . 761

7.9. Immobilizing MembraneProteins onto Solid Supports . . . . 762

7.10. Folding Membrane Proteinsfrom a Denatured State . . . . . . . . . 763

7.11. Expressing Membrane Proteinsin Cell-Free Systems . . . . . . . . . . . . 765

8. CONCLUSION:OPPORTUNITIES VERSUSCONSTRAINTS. . . . . . . . . . . . . . . . . . 766

Detergent:a surfactant with theability to solubilize fats

MP: membraneprotein

1. INTRODUCTION: STRANGERIN A STRANGE LAND

Thirty-five years have elapsed since the art ofusing detergents to handle membrane proteins(MPs) emerged from the “cooking recipe” ageand entered that of physical chemistry (1, 2).Yet, most biochemists will confess to a feel-ing of nervousness when compelled to deal

with membrane-associated proteins. MPs in-deed have earned a well-deserved reputation forbeing hard to handle once extracted from theirnatural environment and made water soluble,and the search for the detergent and conditionsthat will confer upon them a modicum of stabil-ity is known to be time-consuming and, moreoften than not, frustrating.

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Outsidemedium

Periplasm

a b

2.5 nm

Octyl-β-D-glucoside

OmpF

Figure 1How surfactants associate with a membrane protein. (a) Space-filling model of Escherichia coli’s trimeric outer membrane proteinOmpF [Protein Data Bank (PDB) accession code 2OMF; see Reference 154]. Hydrophobic and aromatic amino acid side chains, shownin black, form an ∼2.5-nm wide belt, which, in situ, faces the hydrophobic interior of the membrane (approximated by horizontallines). Reprinted from Reference 11. (b) The distribution of the detergent octyl-β-d-glucoside (purple cage) around OmpF (in skeletonrepresentation), as determined by neutron crystallography, closely follows the belt of hydrophobic residues. Adapted from Reference 155.

A glimpse at Figure 1a makes it immedi-ately obvious why integral MPs—the only oneswe are concerned with in this review—cannotbe handled in pure water: The part of their sur-face that, in situ, is in contact with lipid acylchains and/or the transmembrane surface ofother proteins is, as a rule, highly hydrophobic.A solution of MPs in an aqueous buffer doesnot stay monomeric because the hydrophobiceffect, which tends to minimize the number ofwater molecules in contact with apolar surfaces(3), will drive MP transmembrane surfaces tointeract with one another. This results in aggre-gation and, most often, in precipitation. Addingsurfactants to the solution prevents this phe-nomenon. Surfactants are molecules that com-prise at least one polar and at least one apolarmoiety. In aqueous solutions, the polar groupsare readily solvated, whereas the apolar onesare pushed toward the air-water interface fromwhich they displace water molecules. This re-duces the free-energy cost of creating this in-terface, which lowers the surface tension, hencethe name surfactant.1 Surfactants also displace

1Most proteins are surfactants, but they are not included un-der this term in the present text.

Integral membraneprotein: a proteinthat is in contact withthe hydrophobicinterior of a biologicalmembrane

Surfactant: acompound that lowersthe surface tension ofwater

water from the hydrophobic surface of MPsonto which they adsorb, making it more polar.If their concentration is high enough, they mayform a continuous belt that covers what, in situ,was the transmembrane surface (Figure 1b).The resulting complex can be readily soluble.For the protein to remain monomeric, surfac-tant/protein interactions must overcome pro-tein/protein ones, a process that depends bothon the properties of the surfactant and on itsconcentration.

Before being handled in aqueous solutions,MPs must be extracted from membranes. Thatis, the protein-lipid and protein-protein inter-actions that anchor them in the membrane mustbe replaced with protein-surfactant ones. Thisis usually achieved using a special class of sur-factants that biologists call detergents (phys-ical chemists hate the term, which they re-serve for laundry). Detergents are surfactantswhose solution properties allow them to dis-perse fats and other hydrophobic molecules.In contrast to the lipids that comprise biologi-cal membranes, which, as a rule, form bilayers,detergents self-organize in water in the formof small aggregates, comprising typically 40–100 molecules, called micelles. Their apolar

www.annualreviews.org • Nonconventional Surfactants 739

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Critical micellarconcentration (cmc):the concentration of asurfactant above whichits molecules start toorganize into micelles

moieties are grouped in the core of the micelle,away from water, and the polar groups face thesolution. The reason why biological lipids formextended planar structures and detergents formsmall closed ones is geometric: In projection onthe plane of the membrane, the polar and apolarmoieties of lipids occupy comparable areas—that is, on average, lipid molecules look moreor less cylindrical—so that the juxtaposition ofmany molecules forms a flat monolayer, two ofwhich appose to form the membrane. In de-tergents, the apolar part is less bulky than thepolar one, which bends the interface, generat-ing spheres, ellipsoids, or cylinders, called mi-celles (3, 4). The concentration above which mi-celles form—the critical micellar concentration(cmc)—depends on a balance between the hy-drophobic effect and translational entropy, thefirst effect driving detergent molecules to as-semble, whereas the second one favors their dis-persion. Micelles are in rapid equilibrium withmonomers (see, e.g., Reference 5). For straight-forward thermodynamic (entropic) reasons, theformation of micelles essentially buffers theconcentration of monomers at the cmc, so thatevery new addition of detergent to the solu-tion results in the formation of more micelles.Above the cmc, the chemical potential of the de-tergent remains almost constant (3). Detergentsdissolve hydrophobic or amphipathic moleculesthat partition into micelles.

Because they partition efficiently intomembranes while having a molecular shapedifferent from that of lipids, detergents stressthe membrane/water interface, which theytend to bend. Ultimately, if the detergent is“strong” enough, the planar structure becomesenergetically unfavorable, holes form, andmembrane constituents become dispersed intomixed micelles comprising detergent, lipids,and MPs. For a detergent to be solubilizing,the membrane has to break up before micellesof pure detergent appear in the solution. Ifsuch is not the case, membranes into whichsome detergent has partitioned will coexistwith micelles. This happens with some “weak”detergents and with non-detergent surfac-tants. For detergents to efficiently resolve a

membrane into its constituents, pro-tein/detergent and lipid/detergent interactionshave to overcome the protein/protein, pro-tein/lipid, and lipid/lipid interactions that keepthe membrane together. Detergents used tosolubilize biological membranes are, therefore,out of necessity dissociating.

It has long been observed that, once exposedto detergents, most MPs rapidly lose their func-tionality (for two examples among hundredsof studies, see, e.g., References 6 and 7). Whythis is so is seldom studied in detail, is probablyvariable from one protein to the next, and isnot the object of a consensus among membranebiochemists (for reviews, see, e.g., References8–11). An extensive discussion of this matteris beyond the scope of the present review, andI present only my own views about it. Muchof the data that my coworkers and I havecollected in the course of more than 30 yearsof work with half-a-dozen MPs suggests thata major contribution to the destabilization ofMPs by detergents is the dissociating prop-erties of the latter, i.e., their ability to disruptprotein/protein and protein/lipid interactions,a property that is the very reason why theyare used in the first place. Protein/protein andprotein/lipid interactions, however, are essen-tial to MP stability: Oligomeric MPs usuallyfeature subunit/subunit interactions in thetransmembrane region; the three-dimensional(3D) structure of monomeric MPs, as well asthat of subunits, depends on protein/proteininteractions between their transmembranesegments; and most MPs are extracted frommembranes along with bound lipids, whichstabilize them. Detergents compete with all ofthese interactions, and micelles act as a “hy-drophobic sink” for molecules that, initially,were associated with the MP under study.Delipidation is one of the most, if not themost, common causes of MP inactivation. Thedestabilizing effect of diluting lipids, subunits,and/or hydrophobic or amphipathic cofactorsamong detergent micelles explains the factthat, for a given detergent, working close tothe cmc, i.e., in the presence of few micelles,will often improve the stability of MPs.

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The obvious underlying mechanism is thatreducing the concentration of micelles limitsthe hydrophobic volume into which lipids,cofactors, and subunits can diffuse or, statedanother way, increases their concentration inthe mixed micelles. This favors their stickingtogether rather than coming apart. An equallyfrequent observation is that supplementingan MP in detergent solution with lipids veryoften will stabilize it. This can be due to thepreservation of interactions with the lipids thathelp the protein to keep its 3D structure and/orto the lipids preventing access of the detergentto vulnerable spots at the transmembranesurface. Other causes of destabilization canalso be considered. For example, it can beargued that the dynamics of transmembraneα-helices within helix bundles is restrictedby the geometry of the bilayer more thanit is within a detergent micelle or that thelateral pressure gradient within the bilayer isimportant for their stability (for a discusssion ofthese effects, see Reference 12). Our own workhas been based on the belief that finding waysto limit the disruption of protein/protein andprotein/lipid interactions would be a decisivestep toward improving MP stability.

2. KEEPING MEMBRANEPROTEINS WATER SOLUBLE INTHE ABSENCE OF DETERGENTS

Because dealing with solubilized MPs is neces-sary to understanding their structure and func-tion and because most MPs become unsta-ble once solubilized, many attempts have beenmade to develop countermeasures. One ap-proach is to make the protein itself more stable,either by selecting it from appropriate organ-isms, e.g., hyperthermophiles, or by engineer-ing it. The latter strategy has led to remarkablesuccess (see, e.g., References 13–16), and it cer-tainly holds great promise for the future.

An alternative or complementary approachis to make the environment less destabilizing.After being extracted from their originalmembrane environment, MPs traditionallyare kept water soluble with a detergent.

Fluorinatedsurfactant (FS):a surfactant whosehydrophobic chain isfluorinated

Amphipol (APol): anamphipathic polymerthat can keepmembrane proteinswater soluble indetergent-freesolutions as smallindividual entities byadsorbing onto theirtransmembranesurface

Bicelle: a patch ofbilayer whose rim isstabilized by smallsurfactants such asDHPC

Investigators may resort to the same detergentthat was used for extraction, which theygenerally use at a lower concentration, soas to limit MP destabilization. Alternatively,MPs, once solubilized, are often transferred toanother less aggressive detergent, a detergentmixture, or a lipid/detergent mixture. Thereis indeed no fundamental necessity to use astrongly dissociating detergent once a proteinhas been extracted. Weak detergents, such asdigitonin or surfactants of the Tween series,have long been used for this purpose, despitetheir chemical heterogeneity and less than sat-isfying micellar properties (very low cmc, largemicelles). Once the protein has been extracted,however, what the biochemist needs is to keepit soluble and to prevent it from aggregating,conditions that can be provided by surfactantsthat are not necessarily able to solubilizemembranes. This has led to the developmentof such molecules as tripod amphiphiles(17–19), surfactants with rigid hydrophobictails containing cycles (20), peptitergents (21),lipopeptides (22, 23), peptergents (24), fluori-nated surfactants (FSs) (25, 26), or amphipathicpolymers called amphipols (APols) (27), mostof which are not good membrane solubilizers(for brief overviews, see References 11 and 28).A major part of this review is devoted to thelatter two approaches. Earlier reviews on APolsand FSs are found in References 11 and 28–30.

Because replacing biological lipids withother surfactants is generally detrimental to MPstability, an obvious alternative is to reinsertthem, after extraction, into a lipid environment.This may seem self-defeating as the lipids anMP is normally in contact with generally or-ganize into bilayers, which can be dispersed asvesicle suspensions but do not form the smallentities most suitable for biochemical and bio-physical approaches. Bilayers can, however, befragmented into small patches by mixing lipidswith certain surfactants, e.g., bile salts or short-chain lipids such as dihexanoylphosphatidyl-choline (DHPC), which tend to segregate fromthe surfactant-saturated lipid bilayers and formthe rim of small lamellar discs called bicelles.MPs inserted into these discs find themselves


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Nanodisc (ND): asmall patch of bilayerstabilized byamphipathic rimproteins

Nonconventionalsurfactant (NCS): asused in this text, eithera nanodisc, anamphipol, or a(hemi)fluorinatedsurfactant

Membranescaffolding proteins(MSPs): nanodisc rimproteins derived fromapoA-1

surrounded by a bilayer-like environment.Bicelles are a highly interesting medium thathas been used, among other things, for MPNMR studies (31–36), as well as for MP crys-tallization (37, 38). For want of space, bicellesare not discussed here.

Over the past decade, a variant of bi-celles has been actively investigated, the rimof which is formed by amphipathic proteins.These structures, which can integrate MPs upto a certain size determined by the structureof the rim proteins, are variously called nan-odiscs,2 nanolipoprotein particles, nanoscaleapolipoprotein-bound bilayers, or reconsti-tuted high-density lipoprotein particles, here-after lumped under the collective name of nan-odiscs (NDs). NDs have been the object of tworecent reviews (39, 40). This article includes abrief description of what NDs are and whichMP studies have been done thanks to themand also compares their prospects with thoseof APols and FSs.

Throughout this text, APols, NDs, and FSsare collectively referred to as nonconventionalsurfactants (NCSs).

3. PRINCIPLE AND MOLECULARORGANIZATION OFNONCONVENTIONALSURFACTANTS

NDs, FSs, and APols have quite different chem-ical compositions and molecular structures,which are reflected in the very distinct waysthey assemble into particles when they are dis-persed in aqueous solutions. This, in turn, hasprofound effects on the nature and propertiesof the complexes they form with MPs.

3.1. Nanodiscs

NDs were introduced by S.G. Sligar (Uni-versity of Illinois) and his coworkers as thespin-off of a very large body of work onhigh-density lipoproteins (for a review, seeReference 39). Small NDs consist of a patch

2The name NanodiscTM is a trademark of Nanodisc Inc.

of, typically, 130–160 lipids, organized as abilayer and surrounded by stabilizing proteins.The latter are often the so-called membranescaffolding proteins (MSPs), which are derivedfrom human high-density lipoprotein apoA-1by modifications such as pruning away someundesired domains (41) and duplicating otherdomains so as to increase the protein’s lengthand, thereby, the perimeter of the ND (42).MSPs can also be endowed with a polyhistidinetag (42). Other proteins can be used as well(see References 43–45). The structure ofNDs, whether empty or containing MPs, hasbeen intensely studied by such approaches assmall-angle X-ray scattering (SAXS), atomicforce microscopy (AFM), size exclusion chro-matography (SEC), native gel electrophoresis,electron microscopy (EM), solid-state NMR(ssNMR), Fourier-transform infrared spec-troscopy (FTIR), and various other types of op-tical spectroscopy (reviewed in References 39,40). The thickness of an empty ND is that of abilayer (42, 46, 47). The size of the disc dependson the rim proteins, and for a given protein thenumber of encapsulated lipids depends on thesurface area of the lipids (46). Overall diametersreported to date vary between ∼10 and ∼20 nm(reviewed in References 39, 40). How variableit is from disc to disc in a given population isunder investigation (see Reference 47, and thereferences therein). The molecular mass of thesmallest NDs is ∼150 kDa. ND lipids undergophase transitions similar to those of extendedbilayers, although, for entropic reasons, thetransitions are broader, and the behavior of thefirst two rows of lipids in contact with the rimproteins is altered (48, 49).

Original versions of NDs contained twocopies of MSP per disc, but these can alsobe fused one to another, resulting in objectscontaining a single protein per disc (42).Various models have been proposed for thearrangement of the MSPs in NDs, and ex-tensive molecular dynamics (MD) simulationshave been carried out. Most data, particularlyssNMR determination of the conforma-tion of specific classes of MSP amino acidresidues (50) and MD simulations (reviewed in

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5 nm

a

db

c

Carboxyl oxygen

Octylamide side chain

Isopropylamide side chain

Water (within 0.5 nm ofparticle center)

Monomers of membranescaffold protein

Phospholipidbilayer

h

Figure 2Molecular dynamics (MD) structures of nonconventional surfactants. (a, b) MD structure of a nanodisc.Model viewed (a) perpendicular to the bilayer and (b) in the plane of the bilayer, based on the molecular beltmodel of discoidal high-density lipoprotein (156). Two monomers of the membrane scaffold protein form anamphipathic helical belt around a segment of phospholipid bilayer. Model by S.C. Harvey from Reference39. (c, d ) MD structure of an amphipol (APol) particle. A snapshot from the end of a 50-ns MD simulation ofa fully hydrated A8-35 particle. The particle (Rg ≈ 2.4 nm) consists of four identical 10-kDa A8-35molecules, whose side chains were distributed randomly along the backbone. In panel c, groups are coloredas described in the key, with black lines indicating the polyacrylate backbone. Surrounding waters,hydrogens, and ions have been excluded for clarity. In panel d, only the octylamide side chains, backbone(black lines), and water within 0.5 nm of the particle center are shown in order to highlight the formation ofsubmicellar domains and a hydrated particle core. The scale bar represents ∼5 nm ( J.N. Sachs, personalcommunication). Nanodiscs and APols are represented approximately to scale.

Reference 51), favor the “molecular belt”model in which the protein (or proteins) wrapsaround the rim of the disc (Figure 2a,b)(reviewed in References 39, 40, 51).

NDs have two unique properties comparedto the two other systems discussed in this re-view. First, the MPs they harbor find themselvessurrounded by a medium that is extremely sim-ilar to a normal bilayer. MP functions that de-pend, in one way or another, on interactionswith lipids therefore stand a better chance tobe faithfully reproduced than in APols or FSs.Second, the defined size of the rim proteinssets that of the disc and, therefore, the dimen-sions of the MP or MP complexes that can beencapsulated. In contrast to detergents, APols,

or FSs, MSPs cannot accommodate MPs be-yond a certain size, and the probability thatdiscs can fuse, allowing MPs trapped in dif-ferent discs to temporarily occupy the samedisc, seems remote. NDs can be used, there-fore, to investigate issues where the degree ofassociation of MPs with one another is of pri-mary importance (Section 7.3). The flip side, ofcourse, is that, as of now, MPs with large trans-membrane domains cannot be handled in NDs(Section 5.2).

3.2. Fluorinated Surfactants

The chemical structure of FSs resembles thoseof classical detergents, but their hydrophobic


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tails contains fluorine atoms (Figure 3). Therationale behind the use of such compoundsis based on the poor miscibility of alkanes andperfluorinated alkanes (52–55). FSs indeedare not cytolytic (they are not detergents)

H

FF FF FF

FF

R = F

R = C2H5

R = C2H5

R = C2H5

F-TAC

HF-TAC

DPn = n = 5-8

FF FFO NH

n

R

OH

OHHO

S

FF FF FF

FF FF FF

RS

O

O

OH

OH

HO

HO

O

R1O

R1 = R2 = H

R1 = R2 = β-D-Glu

R1 = β-D-Glu R2 = H R = F F6-DiGluH2F6-DiGlu

F6-TriGluH2F6-TriGlu

R = F

R = F F6-MonoGlu

R2ONH

HN

HN

OO O

NiN

O

O

OOH2

OH2

O

FF FF FF

FF FF FFO NH

HN

yxR

OH

OHOH

S

a

b

c

d

e

S

FF FF

FF FF FF

FF–

H

O NH

OHOHOOG488

R = C2F5

R = F

R = C2H5

y = 4.9

y = 4.94

y = 8

x = 0.1

x = 0.06

x = 2

O

NH N

+F(CF2)p(CH2)m

(CH2)3 (CH2)3 SO3–

CH3H3C

because they do not partition well into lipidmembranes (25, 55–61). Because they arenot detergents, FSs originally attracted littleattention from membrane biochemists (see,e.g., References 25, 62, 63), with the exceptionof perfluorooctanoate, which has been usedfor MP electrophoresis (64, 65). It couldnevertheless be hoped that FSs might be lessprone than detergents to destabilize MPs forthe following two reasons: (a) Lipids, subunits,and hydrophobic cofactors should partitionless favorably into FS micelles; and (b) becausefluorinated alkyl chains are more bulky andmore rigid than alkanes and have little affinityfor hydrogenated transmembrane proteinsegments, they ought to be less efficientthan detergents at disrupting protein/proteininteractions. By the same token, however,FSs bearing perfluorinated chains could beexpected to be ineffective in preventing MPsfrom aggregating, which early data seemed tobear out (25, 63). To try to improve interactionswith the methyl group-covered transmembranesurfaces of MPs while preserving the overalllyophobic (“lipid-hating”) character of FSmicelles, a hydrogenated tip was grafted ontothe fluorinated tail, yielding “hemifluorinatedsurfactants” (HFSs) (Figure 3). Hereafter, FSsand HFSs are collectively referred to as (H)FSs.

The development of (H)FSs, which hasgone through three main phases, has beencarried out through a long-term collabo-ration between our laboratory and that of

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Chemical structure of some fluorinated surfactants.(a) F-TAC (C8F17-C2H4-S-poly-tris-(hydroxy-methyl)aminomethane) (see References 25, 56, and57) and HF-TAC [C2H5-C6F12-C2H4-S-poly-tris-(hydroxymethyl)aminomethane] (see References 26and 55). (b) (H)F-Mono-, Di- and TriGlu; F6- andH2F6-, as used in the text and in Figure 7, refer tohydrophobic moieties, where R = F and R =C2H5, respectively. See References 71 and 72.(c) Phenyl-HF-NTANi. See Reference 138.(d ) (H)F-TACs labeled with Oregon Green(OG448). See Reference 61. (e) A fluorinatedamidosulfobetaine, FASB-p,m. See Reference 74.

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B. Pucci (University of Avignon). The firstmolecules to be tested had as a polarhead group a short hydrophilic oligomer de-rived from tris-(hydroxymethyl)aminomethane(THAM) and a perfluorinated hydrophobicmoiety (Figure 3a) (25, 56, 57). In a secondstage, the tail was hemifluorinated (Figure 3a)(26, 57, 66). These molecules yielded verypromising results, and most of the appli-cations of (H)FSs that have been exploredto date have been developed using them(Section 7). The oligomeric polar head of (H)F-TAC—where “(H)F-” refers indifferently tothe fluorinated or the hemifluorinated form—however, has been a concern from the startbecause it is chemically polydisperse. Fromthe biochemist’s point of view, this means that(H)F-TAC batches consist of a mixture ofmolecules with slightly different properties andthat this mixture will never be exactly the samefrom one batch to the next.

Replacing the poly-THAM oligomer witha monodisperse headgroup initially seemeda straightforward proposition, which was ex-plored as a third stage of development. Thisstep turned out to be highly frustrating. In-deed, the surfactants obtained upon graft-ing (hemi)fluorinated chains onto monodis-perse polar heads that, when associatedto alkyl chains, yield efficient detergents,such as an aminoxyde (67), a monodispersepolyethyleneglycol group (see Reference 28), orsaccharidic groups derived from galactose (25,68), lactose (69), or maltose (70) featured unsat-isfactory properties. A recurrent problem wasthat most of the molecules thus obtained tendedto form huge polydisperse micelles by them-selves and highly polydisperse MP/(H)FS com-plexes (for a discussion, see Reference 71). Thisbehavior suggested that the bulky hydropho-bic moiety of (H)FSs requires a bulkier hy-drophilic head than classical detergents do tocreate the overall molecular asymmetry thatleads to the formation of small globular mi-celles (4). A systematic investigation was, there-fore, undertaken in which polar heads carry-ing one, two, or three glucose moieties weregrafted onto perfluorinated, hemifluorinated,

Lyophobic: havinglittle affinity for lipids;perfluoroalkanes areboth hydrophobic andlyophobic

Hemifluorinatedsurfactant (HFS): asused in this text, asurfactant with afluorinatedhydrophobic chainthat ends with ahydrogenated tip

(Hemi)fluorinatedsurfactant,abbreviated (H)FS:is used whenever thematter underdiscussion appliesindifferently to FSsand HFSs

THAM: tris-(hydroxy-methyl)aminomethan

(H)F-TAC: an (H)FSwith an oligomericpolar head derivedfrom THAM

or hydrogenated hydrophobic chains (72). Thisstudy led to the identification of two chemicallydefined (H)FSs, F6-DiGlu and H2F6-DiGlu(Figure 3b), which, as described below (Sec-tions 5.3 and 6.2), form with MPs small, well-defined complexes in which MPs are stabilizedas compared to detergent solutions (71).

A new kind of hemifluorinated surfactant hasrecently been introduced in which the tip ofthe hydrophobic chain is perfluorinated, but amore or less extended hydrocarbon region isinserted between it and an amidosulfobetainepolar head (Figure 3e) (73, 74). To avoid con-fusion, these compounds are designated here bythe name used by their developers, FASBs (fluo-rinated amidosulfobetaines). As (H)FSs, FASBsdo not by themselves extract MPs (74).

3.3. Amphipathic Polymers(Amphipols)

Detergents and FSs are in a constant equilib-rium between monomers, micelles, and the sur-factant layer that covers the transmembrane re-gion of the protein and makes it hydrophilic.MPs will aggregate if the surfactant concen-tration drops below its cmc, meaning thatMP/detergent complexes must be handled inthe presence of free micelles. The initial ideabehind the concept of APols was to designmolecules that would have such a high affin-ity for the surface of the protein that traces offree surfactant in the solution would suffice tokeep the protein soluble. An MP transferredto such a medium would face no difficulty re-taining its associated lipids, cofactors, and/orsubunits and, therefore, should be strongly sta-bilized. This concept led my colleagues C.Tribet and R. Audebert (ESPCI, Paris) and my-self to devise a family of short amphipathic poly-mers that carry a large number of hydrophobicchains and thus can associate with the trans-membrane surface of MPs by multiple contactpoints. This was expected to result in a vanish-ing low rate of spontaneous desorption and avery high affinity for MP transmembrane sur-faces. The new molecules were dubbed am-phipols (APols) to distinguish them from other


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O

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Figure 4Chemical structures of amphipols (APols). (a) A8-35, a polyacrylate-basedAPol. See References 27 and 76. (b) C22-43, a phosphorylcholine-based APol.See References 82 and 85. (c) A sulfonated APol. See Reference 84. (d ) Aglucosylated, nonionic APol (NAPol). See References 87–89.

amphipathic polymers, such as those used in theindustry to stabilize dispersions of mineral par-ticles or droplets or to control the rheology ofsolutions.

For an amphipathic polymer to form com-pact and stable complexes with an MP, the dis-tribution of its hydrophobic chains must bedense, its solubility in water must be high, andit must be highly flexible. It is also desirable thatthe synthesis of tens of grams be reasonably sim-ple and reproducible. A first series of APols wasdesigned in 1994. One of its members, A8-35(27), has since become by far the most exten-sively studied APol (75, 76). A8-35 (Figure 4a)is composed of a relatively short polyacrylatechain (∼70 residues), in which some (∼17) ofthe carboxylates have been grafted at randomwith octylamine and some (∼28) with isopropy-lamine. The ∼25 free acid groups are charged inaqueous solutions (75), which makes the poly-mer highly water soluble, whereas the octy-lamide moieties, which are spaced along thechain about every nanometer (statistically), ren-der it highly amphipathic. The incorporationof isopropylamide groups is not essential: In-deed, the sister structure, A8-75, which doesnot contain any such groups, is just as goodas A8-35 when it comes to keeping MPs wa-ter soluble (27, 77). However, A8-35 features alower charge density along the chain than A8-75, which seems to have a favorable effect on thestability of MPs ( J.-L. Popot and coworkers,unpublished observations). The average molec-ular weight of a molecule of A8-35 is 9–10 kDa.Batches of A8-35 are a complex mixture ofmolecules with a variable overall length and avariable distribution of lateral chains. Whetherthis heterogeneity represents a favorable or anunfavorable feature from the biochemist’s andbiophysicist’s points of view remains an openquestion because it is not known to which ex-tent the protein may select among the vari-ety of chains offered to it. It is worth not-ing, however, that A8-35 batches with a muchnarrower length distribution did not appearto behave differently from more polydispersebatches (C. Tribet & F. Giusti, unpublishedobservations).

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A8-35 is highly water soluble (>200 g.L−1).In a process that is rather unusual for amphi-pathic polymers, it forms well-defined, smallglobular particles in aqueous solutions (76).Each A8-35 particle has a mass of ∼40 kDa.It comprises, therefore, slightly more thanfour average A8-35 molecules and a total of∼80 octyl chains. The latter number is closeto that of hydrophobic chains in a typicaldetergent micelle. Forster resonance energytransfer (FRET) studies of mixtures of A8-35molecules labeled with pairs of complemen-tary fluorophores have shown that their criticalaggregation concentration (that above whichindividual molecules start self-assembling) is<2 mg.L−1 (F. Giusti, J.L. Popot & C.Tribet, unpublished observations), meaningthat, under most usual conditions, nearly all ofthe free polymer is assembled into particles. AnMD model of an A8-35 particle is shown inFigure 2c,d ( J. N. Sachs, personal communi-cation). Its calculated radius of gyration, Rg ≈2.4 nm, is identical to that measured experimen-tally (76). An unexpected feature of the MDmodel is the tendency of water molecules tooccupy the center of the particle (Figure 2d ).There is also a marked tendency for octyl chainsto form submicellar clusters in which octylchains belonging to distinct APol moleculesclump together (Figure 2d ).

Variations around the chemical structure ofA8-35 have been experimented with. The orig-inal study included molecules that were longerthan A8-35 and/or carried a higher charge den-sity (27), some of which were used in subsequentworks (77, 78, 79). Related structures have beenproposed by others (80, 81). One of the con-straints imposed by the chemical structure ofA8-35 is that its solubility is provided by car-boxylate groups. For this reason, it cannot beused below pH 7 (75, 76, 82, 83) nor in thepresence of mM concentrations of Ca2+ (82,84). These limitations have prompted the de-sign of APols that are zwitterionic (Figure 4b)(81, 82, 85), sulfonated (Figure 4c) (84), ornonionic (Figure 4d )—the latter also thefruit of a long-term collaboration between our

NAPol: nonionicamphipol

laboratory and that of B. Pucci (86–89). Allof these structures have proven able to trapMPs and, generally, have been found to be pHand calcium insensitive. Charged, amphipathicderivatives of pullulane (90), by contrast, turnedout to be very inefficient at keeping MPs sol-uble (91). Most of the background studies anddevelopments to date have been carried out us-ing A8-35, but as discussed below (Section 7),there are some applications for which A8-35 isnot the most suitable APol or cannot be usedat all, making it highly desirable to develop andvalidate alternative APols.

Because APols are relatively large molecules,grafting a small functional group onto them willgenerally not affect their solution properties.A8-35 has thus been derivatized with biotin (92)or with various fluorophores (93; F. Giusti, un-published data), opening the way to many inter-esting experiments (Sections 5.1 and 7.9). Thepolymerization process used for the synthesisof nonionic APols (NAPols), known as telom-erization (87, 88), makes them easily amenableto stoichiometric functionalization with a sin-gle group per chain. A8-35 has also been la-beled isotopically with 14C (77), 3H (83), or 2H(75). Deuteration has been particularly usefulfor NMR (94, 95) and neutron scattering (75,76, 83) studies.

4. TRANSFERRING MEMBRANEPROTEINS TONONCONVENTIONALSURFACTANTS

Although direct extraction of MPs by APolshas been observed occasionally (reviewed inReference 29), APols, NDs, and (H)FSs arenot normally used to extract MPs. The usualprocedure is to transfer the MP to the NCSfollowing solubilization with a nondenaturingdetergent. In the case of NDs and APols,scaffolding proteins and lipids or the polymer,respectively, are usually supplied to the proteinin detergent solution. Upon detergent removal,the systems self-assemble (see, e.g., References43, 44, 46, 83, 93, 94, 96). The ratio of NDs


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to MP can be adjusted so as to facilitate thetrapping of oligomers or, on the contrary, tofavor that of monomers (Section 7.3). WithAPols, an excess of polymer with respect towhat the MP actually binds is necessary toobtain homogeneous MP/APol complexes,so that, after trapping, part—typically abouthalf—of the APol is protein bound and part ispresent as free APol particles. Lipids, if retainedby the protein or if present in the detergentsolution, will be trapped along with the protein,forming ternary MP/lipid/APol complexes,which may be important for maintaining thestability and function of the protein (see, e.g.,References 83 and 97–99 and Section 6.1). Itis of great interest that complex mixtures ofdetergent-solubilized MPs, such as the wholesupernatant obtained after solubilizing a bio-logical membrane preparation, can be trapped,whether with NDs or with APols, in the formof discrete MPs or MP complexes, which canthen be separated, e.g., by centrifugation insucrose gradients, SEC, or isoelectrofocusing(IEF) (cf. Section 7.4) (89, 100).

Similar transfer procedures can be used for(H)FSs, but what has often been done in thecourse of developing these compounds has beento layer a ternary MP/detergent/(H)FS mix-ture on top of a sucrose gradient containingthe (H)FS to be tested (25, 26, 71). Upon ul-tracentrifugation, MP/(H)FS complexes enterthe gradient, leaving the detergent behind them(see below, Section 6.2). This procedure has theadvantage of consuming relatively little (H)FS,as compared, for instance, to dialysis or molec-ular sieving, which is an important criterionwhen working with compounds whose synthe-sis is difficult and whose availability is generallylimited.

As described below, MPs are not alwaystransferred to NDs, APols, or (H)FSs fullyfolded from a solution in nondenaturing de-tergent: They can be directly folded in NCSs,either starting from the full-length, denaturedprotein in sodium dodecyl sulfate (SDS) or urea(Section 7.10) or in the course of in vitro cell-free protein synthesis (Section 7.11).

5. STRUCTURE OF MEMBRANEPROTEIN ANDNONCONVENTIONALSURFACTANT COMPLEXES

The composition, size, homogeneity, structure,and dynamics of MP/NCS complexes havebeen closely scrutinized because they largelydetermine the suitability of the complexes forvarious studies.

5.1. Membrane Protein/A8-35Complexes

A8-35 has been shown to form a complex with,and maintain solubility of, a very broad range ofMPs. Those include all of the 30-odd integralMPs that have been tested to date. Theirmolecular masses range from <5 kDa (a singletransmembrane α-helix) to >1.1 MDa [mito-chondrial complex I, which probably harbors∼70 transmembrane α-helices (101)], and theyrepresent any conceivable diversity of origins,functions, and structures (for a review, seeReference 29). A8-35 has recently been usedto trap and purify mitochondrial supercomplexB (T. Althoff & W. Kuhlbrandt, personal com-munication; see Section 7.7), which comprises∼120 transmembrane helices and whose over-all mass is ∼1.7 MDa (102). The interactions ofAPols with other types of objects are beyond thescope of the present review. However, one maymention that APols have been used to stabilizein solution a signal sequence peptide (103),human apolipoprotein B-100 (I. Waldner,A. Krisko, A. Johs, & R. Prassl, unpublisheddata), plant lipid storage proteins (Y. Gohon,personal communication), and semiconductorquantum dots (104, 105). Several studies havebeen carried out of their interactions with lipidvesicles or cells (78, 79, 104, 106). Under mostcircumstances (for some exceptions, see Refer-ence 78), APols will not solubilize preformedbiological or lipid membranes (see Reference29). They can, therefore, be applied to lipidvesicles, black films, or living cells withoutlysing them, which opens up some extremelyinteresting applications (Section 7.8).

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The composition, structure, dynamics, andsolution properties of MP/A8-35 complexeshave been studied by SEC, analytical ultracen-trifugation (AUC), small-angle neutron scatter-ing (SANS), SAXS, FRET, and solution NMR,yielding a rather detailed picture of what thesecomplexes look like. In NMR studies using asmodels the transmembrane domains of outerMP A from Escherichia coli (tOmpA) or Kleb-siella pneumoniae (KpOmpA) and E. coli’s outerMP X (OmpX), each of which consists ofan eight-strand β-barrel, the only detectablecontacts between A8-35 and the protein wereobserved at the barrel’s transmembrane sur-face (Section 7.5). AUC and SANS studies ofbacteriorhodopsin (BR)/A8-35 complexes in-dicate that the polymer layer is compact and1.5–2 nm thick (83), which is only slightlythicker than a detergent layer (107). Measuringprecisely the amount of protein-bound APolis technically difficult, particularly for smallproteins (83, 93). Estimates vary from ∼2 gper g protein for a small, mainly transmem-brane protein like BR (Figure 5c) (83) to∼0.13 g per g for a complex with large ex-tramembrane regions such as cytochrome bc1

(Figure 5a) (D. Charvolin, unpublished data).For large MPs, this corresponds to signifi-cantly less (by 2–3 times) octyl chains than thenumber of detergent molecules bound by thesame protein in detergent solutions (29). Forsmall MPs, the difference is much less (83, 93).In most experiments, such as those by SAXS,SANS, AUC, or solution NMR, MP/A8-35complexes behave like compact, globular par-ticles (83, 94, 108). Upon SEC, tOmpA/A8-35 and BR/A8-35 complexes migrate as thoughthey are somewhat bigger than they actually are(83, 94).

When properly prepared, MP/A8-35 com-plexes are essentially homogeneous (83, 93),although they do not appear as narrowly dis-tributed in SEC as MP/detergent complexes(93). It is, however, difficult to totally avoid thepresence of minor fractions of small oligomers,which can seriously complicate, in particular,radiation scattering experiments (83). Amongthe factors that can lead to polydispersity, if not

tOmpA: thetransmembranedomain of outermembrane protein Afrom Escherichia coli

aggregation, are (a) the use of too little APol atthe trapping step (93), (b) working at pH ≤ 7(82, 83, 94), (c) the presence of Ca2+ ions (82,84, 108), (d ) drifts from the nominal compo-sition of the polymer that result in increasingits hydrophobicity (83, 84), and (e) separatingthe complexes from the free polymer that co-exists with MP/APol complexes at the end ofa trapping experiment (83, 93). The oligomer-ization that follows the removal of free APol isreversible. It has been interpreted as resultingfrom the poor dispersive power of APols: Inthe competition between protein/protein andprotein/APol interactions, the presence of anexcess of polymer favors the formation of MPmonomers, whereas its removal shifts the equi-librium toward aggregation (93). Increasing theionic strength of the solutions progressivelyturns the interactions between cytochromebc1/A8-35 particles from a repulsive to an at-tractive mode (29). In a solution that containsboth protein-bound and free APols, free andbound polymers exchange at the surface of theprotein at a rate that, at least for A8-35, stronglydepends on the ionic strength of the solution(minutes in 100 mM NaCl, hours at low ionicstrength) (93). The underlying mechanism hasnot been studied in detail, but it probably in-volves collisions between MP/APol complexesand free APol particles, followed by fusion, mix-ing, and fission. Such a mechanism is consistentwith the fact that APol particles can deliver reti-nal, a highly hydrophobic cofactor, to refoldedbacterio-opsin (99). By contrast, in the absenceof exchange with another surfactant, APols donot desorb from MPs even under extreme di-lution or extensive washing (29, 77, 93, 109). Athermodynamic analysis of MP/detergent ver-sus MP/APol interactions has led to the conclu-sion that the stability of MP/APol complexesis essentially of entropic origin: When a solu-tion of MP in detergent is diluted below thecmc of the detergent, the release of tens ofdetergent molecules that accompanies the as-sociation with one another of two MPs cre-ates a strong entropic drive toward aggregation;when two APol-trapped MPs aggregate, thedesorption of one or two APol molecules, which


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b a

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Figure 5Models of complexes between membrane proteins (MPs) and amphipols (APols) or nanodiscs (NDs).(a-c) Models of MP/APol complexes. (a) Cytochrome bc1/A8-35 complex (from Reference 29). (b) An “open”model of a complex between the transmembrane domain of E. coli’s OmpA (tOmpA) and A8-35 (fromReference 29). (c) Cross section through a bacteriorhodopsin (BR)/A8-35 complex. Lipids from the purplemembrane, which were retained throughout the solubilization and trapping procedures, are shown in olivegreen (from Reference 83). The volume and distribution given to the APol belts in the three complexes arebased on an ensemble of data including binding, SEC, AUC, SANS, and NMR measurements, obtained oneach individual complex as well as on other MPs (see References 29 and 83 and Section 5.1) Models by D.Charvolin. (d, e) Models of rhodopsin (Rho) (d ) and the serine chemotaxis receptor (Tsr) (e) embedded intoNDs. From Reference 39. The five models are represented approximately to scale.

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associate into particles, is entropically neutral(109).

Although few proteins have been tested yet,it seems that freezing MP/APol complexes isnot detrimental, whereas lyophilizing them canbe (83; Y. Gohon & L. Catoire, unpublisheddata).

In ternary mixtures containing an MP, adetergent, and an APol, the two surfactantsmix at the transmembrane surface of the pro-tein, forming ternary complexes (77, 93, 109).FRET and isothermal calorimetry studies showthat mixing is almost ideal and that the ex-change of one surfactant for the other is isoen-thalpic: Detergents and APols mix freely, andthe composition of the mixed belt of surfac-tant around the MP reflects that of the mixed,protein-free APol-detergent particles (93, 109).The exchange of APol for detergent at the sur-face of an MP is extremely rapid (<1 s) (93).Because APols have no strong preference forthe MP-bound belt versus free APol/detergentparticles, they are easily and rapidly washedaway by an excess of detergent (77, 93, 97,109). Interestingly, MPs can be stabilized bio-chemically even by mixtures of detergent andAPols (97) (see Section 6.1 below). Upon SEC,MP/APol/detergent particles appear more ho-mogeneous in size than pure MP/APol com-plexes (93). These two observations may haveinteresting implications for the choice of crys-tallization conditions (Section 7.6).

5.2. Membrane Protein/NanodiscComplexes

MPs that have been reconstituted into NDs in-clude a number of P450 cytochromes as wellas the NADPH-cytochrome P450 reductase(reviewed in References 39 and 40), BR (110,111), the bacterial Tar chemoreceptor (112), theSecYEG translocon complex (113), and severalG protein–coupled receptors (GPCRs), includ-ing the β2-adrenergic receptor (β2-AR) (114–116), rhodopsin (44, 117), and a μ-opioid re-ceptor (118). The size of the largest transmem-brane domain that can be accommodated into

a ND depends on the rim protein employed.In addition, recent single-particle data suggestthat NDs can have a multimodal distributionof diameters and that MP encapsulation canshift this distribution toward larger diameters(45, 47). The largest system whose encapsu-lation has been reported to date is the BRtrimer, which comprises 21 transmembrane he-lices (111). It is to be expected, however, thatthis constraint will be relaxed as protein engi-neering will produce MSPs and other proteinsable to stabilize larger and larger discs (see, e.g.,References 42, 43, and 45).

MP/ND complexes have been studied ex-perimentally by SEC, SAXS, AFM, EM, circu-lar dichroism (CD), fluorescence spectroscopy,and ssNMR (see, e.g., References 44, 47, 110–112, 115, 117, 118), as well as simulated by MD(119) (for reviews, see References 39 and 40).Essential to the use of NDs for functional stud-ies is the knowledge of the number of copies ofMPs they have captured. Thus, trapping con-ditions have been designed so as to trap ei-ther monomers or trimers of BR (110, 111),monomers of the β2-AR (115) or the μ-opioidreceptor (118), and monomers or dimers ofrhodopsin (44, 117). The Tar chemoreceptorhas been trapped either as dimers or trimers ofdimers (112). Functional studies of biologicalsystems where the number of copies of MP in-volved is a critical factor are one of the mostexciting applications of NDs (see Section 7.3below).

5.3. Membrane Protein/(Hemi)Fluorinated SurfactantComplexes

MP/(H)FS complexes have not yet been stud-ied in great detail, particularly for those (H)FSsthat appear to be most satisfying both from thepoint of view of the chemistry (defined chemicalstructure) (72) and biochemistry (small, well-defined MP/(H)FS complexes) (71). A strikingobservation is that MP/(H)FS complexes mi-grate much faster upon centrifugation in su-crose gradients than MP/detergent complexes


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DM

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DDM F6-TriGlu F6-DiGlu F6-MonoGlu

Figure 6Behavior of cytochrome b6 f in sucrose gradients containing either a detergent, dodecylmaltoside (DDM), orglucosylated fluorinated surfactants at various concentrations. The chemical formulas of F6-Mono-, -Di-and -TriGlu are shown in Figure 3. In DDM, the complex remains primarily a superdimer (D) at aconcentration just above the critical micellar concentration and starts to fragment into monomers (M) athigher concentrations (cf. Reference 7). In F6-TriGlu and F6-DiGlu, it forms well-defined bands of dimerwhatever the surfactant concentration. In F6-MonoGlu, the bands are fuzzy, owing to the polydispersity ofthe complexes. From Reference 71.

(Figure 6) (25, 26, 69, 71). A priori, this couldbe due to an aggregation of the protein and/orto the binding of very large amounts of surfac-tant. However, a detailed study of the complexesformed between cytochrome b6f and a lactose-derived HFS, HF-Lac, has shown that neitheris true: The b6f is dimeric in HF-Lac solutions,as it is in detergent ones as long as it retains itsnative structure (7), and it binds about the samenumber of HF-Lac molecules (∼260) as of do-decylmaltoside ones (69). Instead, it is the muchhigher density of the surfactant [v ≈ 0.6 mL.g−1

for HF-Lac, F6- and H2F6-DiGlu (69, 71)], ow-ing to the presence of the fluorine atoms, whichaccounts for the increased sedimentation coef-ficient of the complexes.

In the course of comparing the behavior of(H)FSs carrying a variable number of glucosemoieties, it was noted that at least two sugarsare needed for (H)FSs to form small globularmicelles and that only in these cases was it pos-sible to obtain well-defined MP/(H)FS com-plexes. By contrast, molecules bearing a singleglucose form long, cylindrical micelles and gen-erate polydisperse MP/(H)FS complexes (71).This is illustrated in Figure 6 in the case ofcytochrome b6 f.

6. FUNCTIONALITY ANDSTABILITY OF MEMBRANEPROTEINS INNONCONVENTIONALSURFACTANTS

The stability and functionality of an MP inaqueous solution is extremely variable, depend-ing both on the protein and on the surfactantthat keeps it soluble, as well as on the con-centration of the latter. Existing data indicatethat, used at comparable ratios of “micellar”(assembled) surfactant to MP, the three typesof NCSs considered here tend to be less in-activating than detergents—that is, they keepMPs from irreversible denaturation for a longertime. Data about the functionality of MPs as-sociated to NCSs, which is a distinct question,are still relatively scarce but, on the whole, veryencouraging.

6.1. Membrane Protein/AmphipolComplexes

Complexation by APols, in most cases, bio-chemically stabilizes MPs as compared to de-tergent solutions (see, e.g., References 27,29, 83, 84, 88, 89, 97, and 120 and the

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references therein). This is illustrated inFigure 7 for the sarcoplasmic reticulum cal-cium ATPase (Figure7a), BR (Figure7b), and aGPCR, the BLT1 receptor of leukotriene LTB4

(Figure 7c). As illustrated by the intermedi-ate curve in Figure 7a, even adding APols toan MP in detergent solution, without remov-ing the detergent or diluting it, may have astabilizing effect. A detailed discussion of themechanisms of MP stabilization by APols isbeyond the scope of the present review. Ex-isting data indicate that the following effectsmay contribute to stabilization: (a) Retentionof MP-associated lipids, cofactors, and subunits(a consequence of reducing the hydrophobicsink, but also of the poorly dissociating char-acter of APols); (b) less efficient competitionwith protein/protein interactions, and (c) damp-ing the dynamics of conformational excursionsof transmembrane α-helix bundles, which lim-its opportunities for unfolding and/or aggrega-tion (for discussions, see References 29 and 84).Stability can be further improved by formingternary MP/lipid/APol complexes (Figure 7c)(97, 120). Although data are still limited, asprinkling of observations suggests that, as is thecase for detergents, NAPols may be even lessdestabilizing than ionic ones are (86; Y. Pierre,unpublished observations).

The functionality of APol-trapped MPs isgenerally preserved (see, e.g., References 29,81, 83, and 98). However, MPs whose func-tional cycle involves large rearrangements ofthe surface of their transmembrane region,as is the case for the sarcoplasmic calciumATPase, may see their activity reversibly slowedor blocked, presumably because the adsorbedpolymer damps such transconformations (fordiscussions, see References 29 and 84). ForBR (83, 99) and, perhaps, the nicotinic acetyl-choline receptor (nAChR) (98), indirect ar-guments suggest that transferring the proteinfrom a detergent to an APol environment fa-vors the rebinding of lipids at the surface of theprotein, which probably contributes to restor-ing membrane-like functionality (Section 7.3).Ligand binding is, very generally, unaffected by

APol trapping (see Sections 7.2, 7.9, and 7.10below).

6.2. MembraneProtein/HemifluorinatedSurfactant Complexes

Following their transfer from detergent solu-tions to (H)FS ones, BR, cytochrome b6 f, thehuman GPCR Smoothened and the mitochon-drial ATP synthase exhibit improved biochem-ical stability (25, 26, 69–71, 121, 122). Such isalso the case of dimers of BLT1 ( J.-L. Baneres,personal communication). The improvement isusually limited when the surfactant concentra-tion is close to the cmc, but it becomes obvi-ous at higher concentrations (Figures 6 and7d ), suggesting that it is mostly a consequenceof the lyophobicity of (H)FS micelles, whichmakes them a poor sink for lipids. BR andb6f are markedly stabilized in mono- and diglu-cosylated (H)FSs, but destabilized in trigluco-sylated ones (71). Although other hypothesescould be considered (see Reference 71), my fa-vorite interpretation of the latter phenomenonis that too large a repulsion between polarheads, which favors the formation of particleswith a small radius of curvature, may tend toeither fragment and/or unfold MPs. The samephenomenon may well contribute to explain-ing the well-known destabilizing character ofcharged detergents. Because (H)FSs with a sin-gle glucose moiety form cylindrical micelles(Section 3.2) and polydisperse MP/(H)FS com-plexes (Section 5.3), and those with three glu-cose moieties are destabilizing, F- and HF-DiGlu (Figure 3b) were identified as optimalamong chemically well-defined (H)FSs (71).

The relative benefits of using either per- orhemifluorinated compounds should be furtherinvestigated. Among the factors to be taken intoconsideration are the following: (a) HFSs aremuch harder to synthesize than FSs; as a con-sequence, they are more costly and availablein more limited quantities; this limitation is ofparticular importance if (H)FSs are to be usedfor MP purification, where large volumes of


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solution above the cmc of the surfactant are re-quired. (b) Initial observations indicating thatFSs are much less efficient at preventing MPsfrom aggregating (25) than HFSs are (26) havenot been supported, or not strongly so, bymore recent experiments, where the difference

appears marginal (71), perhaps because MPpreparations used in more recent studies con-tained less lipids. (c) A difference has been notedbetween the spectra of monomeric BR trappedin either HFSs or FSs: In HFSs, the spectrum ofthe protein resembles that of the native protein;

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Temperature (°C) 45 5550403530252015

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in FSs, it is shifted to the red (“blue BR”) (71).The spectral change results from the protona-tion, owing to a pK shift, of Asp85, a residuethat interacts electrostatically with the proto-nated Schiff base that associates the retinal tothe protein (C. Breyton, unpublished results).This indicates that, directly or indirectly (e.g.,via the lipids), FSs can affect the 3D structure ofthe MPs with which they are complexed. Thefact that BR oligomers are not affected by thiseffect (71) suggests that perhaps this will not bea general concern but may simply reflect eventsoccurring at one specific site at the surface ofone particular protein.

6.3. Membrane Protein/NanodiscComplexes

Because MPs trapped within NDs are not ex-posed to detergents, and because they are sur-rounded by an environment very similar to thenatural one, they can be expected to exhibit im-proved stability. Few studies of this sort seem tohave been reported. Rhodopsin, however, wasindeed found to be much more resistant to ther-mal denaturation in NDs than in detergent so-lutions (Figure 7e) (44).

Functional studies are one of the most in-teresting applications of NDs, and they havebeen pushed much farther than with any ofthe other NCSs considered here. They will bediscussed below along with other applications(Section 7.3).

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 7Stabilization of membrane proteins by nonconventional surfactants. (a) Stabilization of the sarcoplasmic reticulum calcium ATPase byA8-35. The destabilization of the ATPase was initiated by diluting solubilized sarcoplasmic reticulum (SR) into an ethyleneglycol-O,O′-bis(2-aminoethyl)-N, N, N ′, N ′-tetraacetic acid-containing solution, thereby leaving the ATPase in a Ca2+-deprived, solubilizedstate, an environment known to lead to very rapid, irreversible inactivation. (Lower curve) The dilution medium contained 5 g.L−1

(9.3 mM) C12E8; (middle curve) same medium but with the addition of 5 g.L−1 A8-35; (upper curve) same medium as the latter sample,but incubation took place after a 250× dilution with surfactant-free buffer. From Reference 97. (b) Stabilization of BR by A8-35 and bynonionic amphipols (NAPols), as compared to 18 mM octylthioglucoside (OTG). From Reference 89. (c) Stabilization of the LTB4BLT1 receptor by A8-35 and by A8-35/lipid mixtures. (top) Stability upon incubation at increasing temperature; (bottom) stability uponextended storage at 4◦C. From Reference 120. Abbreviations: D+L, detergent + lipids (fos-choline-16/asolectin, 2:1 w/w); AP, A8-35;AP+L, A8-35 + asolectin (5:1 w/w). (d ) Stabilization of cytochrome b6 f by (hemi)fluorinated surfactants as compared to detergents[dodecylmaltoside (DDM) and H-DiGlu; H-DiGlu has the same chemical structure as F- and HF-DiGlu, except for a fullyhydrogenated alkyl chain]. From Reference 71. (e) Stabilization of rhodopsin (Rho) upon integration into nanodiscs (NDs) (in this case,nanoscale apolipoprotein bound bilayers) containing either one or two copies of the protein per disc, versus in rod outer segments(ROS; rhodopsin’s native membrane environment), DDM (29 mM) or octylglucoside (OG; 51 mM). From Reference 44.

7. APPLICATIONS

Owing to space limitation, it is not possible toreview here at length every application that hasbeen explored using each of the three systemsunder discussion. My purpose is, rather, to illus-trate the kinds of studies that may be facilitatedby using one or the other NCSs instead of de-tergents and to provide directions for furtherreading. I will also try, even though I am fullyconscious of the risks inherent in such an exer-cise, to present my feelings about the prospectsand constraints of each of them.

A first benefit of transferring MPs to NCSs isthe stabilization they afford, which may permitthe study, using otherwise classical biochemicalor biophysical approaches, of MPs that wouldbe intractable in detergent solution. What onehas to consider here is whether using NCSsrather than detergents comes at a price and,if so, what price? In addition, NCSs may per-mit studies for which detergents cannot be used(e.g., mediating MP immobilization, definingthe maximal size of the objects trapped, or in-serting MPs into membranes under equilibriumconditions) or are poorly efficient (e.g., MPfolding).

7.1. Constraints forOptical Spectroscopy

A8-35 (93), NAPols (89), and (H)FSs (B. Pucci,unpublished data) do not absorb light signifi-cantly at wavelengths above ∼245 nm. None


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of them fluoresces upon excitation at 280 nm(93; B. Pucci, unpublished data). A8-35 doesnot interfere with CD studies of proteinsdown to at least 180 nm (99, 103, 123, 124;T. Dahmane & F. Wien, unpublished data).(H)F-TACs are not expected to interfere sig-nificantly at these wavelengths. GlucosylatedNAPols and (H)FSs, however, may be expectedto contribute to CD spectra below ∼195 nm,owing to the presence of the sugar residues(125). FTIR studies of MP/A8-35 complexesin the amide region are intractable because ofthe amide bonds of the polymer (Y. Gohon& E. Goormaghtigh, unpublished observa-tions). The same difficulty will arise with sul-fonated APols, NAPols, phosphorylcholine-based APols, and current (H)FSs.

NDs pose a special problem because they in-clude scaffold proteins. It should be possible toreduce the contributions of MSPs by eliminat-ing the tryptophan residues they contain usingsite-directed mutagenesis. The contribution ofMSPs to IR and CD spectra will remain, but itcan conceivably be subtracted.

7.2. Solution Studies of MembraneProtein Mass, Shape, and Interactions

All three NCS systems have been studied bySEC, AUC, SAXS, and/or SANS (see, e.g.,References 71, 83, and 126). A priori, each ofthese techniques can be used to characterizeNCS-complexed MPs. APols and (H)FSs havethe advantages of adding less bulk and com-plexity to the system and of being more eas-ily contrast matched. However, obtaining per-fectly monodisperse preparations of MP/APolcomplexes is challenging, which makes molecu-lar weight and Rg determination of the trappedMP by radiation scattering quite delicate (seeReference 83). Factors that have to be keptin mind when monodispersity is essential havebeen listed in Section 5.1.

APols do not interfere with most of theMP/ligand interactions that have been stud-ied to date (92, 97, 98, 120) (see alsoSections 7.9 & 7.10 below). This does not meanthat one should not be aware of the possibil-

ity of such interferences (for discussions, seeReferences 29 and 92). Thus, preliminary ex-periments with rhodopsin/A8-35 complexessuggested that A8-35 hinders the binding oftransducin and arrestin (see Reference 29). Thispoint has been reinvestigated recently with theleukotriene receptor BLT1. It was observedthat receptor-catalyzed G protein activationwas significantly slower with A8-35-trappedBLT1 than in detergent/lipid mixed micelles.In contrast, when folded in NAPols, BLT1 cat-alyzed GDP→GTP exchange on the Gα i sub-unit with kinetic features similar to those inlipid/detergent mixtures ( J.-L. Baneres, per-sonal communication).

Particularly illustrative of the perspectivesopened by the ND system are studies exploitingNDs to trap monomers or defined oligomers ofvarious MPs. Some of the applications of thisremarkable feature are described in Section 7.3.

Few detailed ligand-binding studies havebeen carried out in (H)FSs yet. The humanSonic Hedgehog receptor Patched was shownby surface plasmon resonance (SPR) to be ableto interact with Sonic Hedgehog after com-plexation by FSs (127). The sensitivity of themitochondrial ATP synthase to the inhibitorsdicyclohexyl carbodiimide and oligomycin wasfound to be higher and to remain more stableover time in (H)F-TACs than it is in detergentsolutions (122).

7.3. Functional Studies

As already mentioned, NDs provide a uniqueapproach to examining the role of oligomer-ization in the biological function of MPs be-cause they make it more straightforward andsafer than any other system to prepare and han-dle monomers and various types of oligomerswhile limiting the risk that transient oligomer-ization complicates the interpretation of the ex-periments. In the case of GPCRs, NDs havebeen used to study the activation of G proteinsby preparations of monomeric β2-AR recep-tor, rhodopsin, and μ-opioid receptor (44, 114,115, 117, 118, 128). Similarly, trapping of singlecopies of translocon in NDs made it possible to

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establish that SecYEG monomers are able tobring about the dissociation of dimeric SecAinto monomers and (pre)-activate the SecAATPase (113). Trapping the Tar chemoreceptoras either dimers or trimers of dimers led to thedemonstration that the formation of the dimersuffices for transmembrane signal transduction(methylation and deamidation), but the super-trimer is required for downstream signaling (ac-tivation of histidine kinase) (112). A vast body ofwork has been carried out on P450 cytochromesand their reductase (reviewed in References 39and 40). They concern, in particular, the ori-gin of cooperativity in ligand binding curvesand the role of lipids and the transmembraneanchor of the cytochromes in P450/reductaseinteractions. NDs have also proven useful instudying the activation of enzymes involved inblood clotting and the role played in it by spe-cific lipids (129, 130). There seems to be littledoubt that NDs will henceforth provide a priv-ileged tool whenever such questions will haveto be dissected.

To date, applications of APols to investi-gations of MP function have mostly focusedon sorting out whether perturbations observedupon MP solubilization are the result of re-moving the membrane environment or of di-rect interference by the detergent. In the caseof the nAChR, it was shown that allosteric equi-libria, which are perturbed upon solubilization,are similar in the postsynaptic membrane andafter trapping with A8-35, indicating that it isthe presence of the detergent, and not the dis-appearance of physical constraints imposed bythe membrane, that is responsible for the per-turbation (98). It was hypothesized that deter-gents might act by displacing lipids from criticalsites at the surface of the nAChR’s transmem-brane domain, where A8-35 would let themrebind. BR, a light-driven proton pump, un-dergoes severe perturbations of its photocycleupon solubilization by detergents. One suchperturbation, the acceleration of the retinal’sSchiff base deprotonation upon capture of aphoton, largely persists after transfer to APols,suggesting that the conformational change thatunderlies this phenomenon results from the

extraction of BR from the purple membranerather than from detergent binding (83). Theend of the cycle, when BR returns to its groundstate, goes back to close to membrane-like fea-tures upon transfer from detergent solution toAPols (83). Comparative studies of the photo-cycle of native BR, trapped in APols along withpurple membrane lipids, and of BR refolded inAPols in the presence or absence of lipids haveled to the conclusion that it is probably the re-binding of lipids at critical sites at the surfaceof the protein upon transfer from detergent toAPols that accounts for the recovery of normalkinetics in the last part of the photocycle (99).

7.4. Proteomics: Isoelectrofocusing,Two-Dimensional Gels,and Mass Spectrometry

Analysis of protein mixtures on two-dimensional (2D) gels usually starts witha separation by IEF followed by electrophore-sis in polyacrylamide gels in the presenceof sodium dodecylsulfate (SDS-PAGE) ina perpendicular direction. IEF of MPs us-ing detergents is notoriously difficult (131).Most APols, which bear net charges, are notcompatible with IEF, but NAPols are. Theresolution of IEF observed with NAPols issimilar to that in neutral detergents (89, 132).Whether the use of NAPols can improve onthe yields obtained with detergents remainsto be ascertained. However, they are certainlyof potential interest as a tool to separate andanalyze fragile MP complexes that do not resistpurification in detergent solutions. (H)FSscould conceivably be used to the same end butare probably at more risk of raising aggregationproblems. FASBs (see Section 3.2) have beenused in 2D electrophoresis as a means to de-tach nonmembrane proteins from erythrocytemembranes prior to solubilizing MPs with aclassical detergent (74). Separating MP/NDcomplexes by IEF may seem a priori an oddproposition because MSPs carry charges.However, provided a constant stoichiometrywith the target MPs is maintained and thelipids are neutral, this should result in a simple


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shift of the isoelectric point of the complex ascompared to that of the MP alone, withoutnecessarily compromising the resolution.

In keeping with their displacement by non-ionic detergents (77, 93, 109), APols are washed

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Figure 8Solution NMR spectra of APol- versus detergent-complexed OmpX.(a) Two-dimensional [15N,1H]-TROSY spectra of [u-2H,13C,15N]OmpXcomplexed by APol A8-35 (left; pH 8.0) or by DHPC (right; pH 6.8) collectedat 30◦C at 700 MHz. (b) A comparison of peak width in the direct dimension.From Reference 108.

away by an excess of SDS. This is reflected inthe fact that, upon SDS-PAGE, MPs migrateat the same position whether they were initiallycomplexed by A8-35 or not (123) and by theabsence of a FRET signal and lack of any CDchange when fluorescent A8-35 is added to anMP in SDS solution as a prelude to renatura-tion (89). In the second dimension of 2D gels,MPs, therefore, separate as they do in the ab-sence of APols, and they can be analyzed bymass spectrometry under the same conditions(132).

Recent data indicate that native MPscomplexed by either A8-35 or NAPols canbe analyzed by matrix-assisted laser desorp-tion/ionization time-of-flight (MALDI-TOF)mass spectrometry, with the APols remainingundetected (95; C. Bechara, G. Bolbach, & S.Sagan, unpublished data).

7.5. Nuclear MagneticResonance Studies

APol-trapped MPs have been studied by solu-tion NMR (94, 95, 108; M. Renault & A. Milon,personal communication). All published studiesto date have been carried out using A8-35, andthey all bear on β-barrel proteins. At this point,the objectives have been mainly to work out thetechnology and to obtain information aboutMP/APol complexes (size, folding state of theprotein, protein/APol interactions, and proteindynamics). Not surprisingly, these studies haveshown that the three proteins studied to date,E. coli’s tOmpA (94) and OmpX (95, 108) andK. pneumoniae’s KpOmpA (M. Renault & A.Milon, unpublished data), are correctly foldedin A8-35 (Figure 8a). Early studies with E.coli’s tOmpA were carried out in the absenceof (ethylenedinitrilo)tetraacetic acid (EDTA)(94). In the presence of EDTA, the resolutionof TROSY-HSQC spectra improves, eventhough the complexes still tumble more slowlythan in DHPC solutions: For OmpX at 30◦C,τ c = 31 ns versus 23 ns (108); for KpOmpAat 40◦C, τ c = 30 ns versus 26 ns (M. Renault& A. Milon, unpublished data). For OmpX,the resulting broadening of the peaks in the

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1H dimension is ∼35% as compared to theirwidth in DHPC solutions (Figure 8b) (108).Solution NMR has been used to map thecontacts between the protein and the lateralchains of the polymer, based on amide 1H peakbroadening in hydrogenated versus deuteriatedA8-35 (94), or on heteronuclear 13C-1H (95),or 15N-edited 1H-1H (M. Renault & A. Milon,unpublished data) nuclear Overhauser effects(NOEs). In all three cases, A8-35 was foundto interact exclusively with the transmembranesurface of the protein. No contacts are ob-served between KpOmpA and the APol’s mainchain (M. Renault & A. Milon, unpublisheddata). Whether contacts with the APol arehomogeneously distributed over the protein’stransmembrane surface and whether the poly-mer’s octyl chains interact with it in the sameway as detergent chains do are still relativelyopen questions (95; M. Renault & A. Milon,unpublished data). Studies of H/D exchangeat amide bonds show that the dynamics andaccessibility of OmpX’s (108) and KpOmpA’s(M. Renault & A. Milon, unpublished data)transmembrane β-strands are similar inDHPC and after trapping with A8-35. It ispossible to determine, by transferred NOEstudies, the structure of ligands bound to largeAPol-stabilized MPs (L. Catoire, unpublishedobservations).

In addition to the slower tumbling rate, aninconvenience that is likely to become less andless relevant as larger MPs are tackled and thetechnology improves, the major drawback ofA8-35 in solution NMR studies is that it doesnot allow one to work at the slightly acidicpH at which the exchange of amide protonswith the solution is slowest. This is of lit-tle import for transmembrane amide protons,which exchange slowly anyway, but it becomescritical for water-exposed protons in the ex-tramembrane loops. This has prompted the de-velopment of alternative, pH-insensitive APols(Section 3.3). Recent data indicate thatTROSY-HSQC spectra can be recorded oftOmpA trapped with sulfonated APols. At pH6.8, it becomes possible to resolve loop amide

protons that are not seen at pH 8.0. Encour-aging data have been obtained recently withNAPols (L. Catoire, personal communication).It is to be expected that further progress in thechemistry of APols and their implementationwill soon make it possible to establish by so-lution NMR the structure of MPs that are toounstable to be studied in detergent solutions.

Solution NMR studies in (H)FS solutionshave not been attempted yet. A potential pit-fall may be aggregation of the protein at thehigh concentrations required by NMR, but theattempt is certainly worthwhile. 19F NMR po-tentially offers some original angles, e.g., to fol-low the distribution of (H)FSs whether in vitroor in vivo.

Solution NMR of MP/ND complexes ishandicapped by the large size of NDs and pos-sible anisotropy problems. Theoretical calcu-lations indicate that the tumbling rate of anempty ND should be comparable to that of an∼200-kDa protein (133). In practice, the res-olution obtained for a 13-residue peptide ad-sorbed onto NDs, although not as good as inbicelles, was somewhat better than expected,probably owing to the peptide moving with re-spect to the surface of the ND. Signal attenua-tion by a spin label dissolved in the lipid phaseyielded information about the secondary struc-ture of the peptide and its arrangement withrespect to the ND surface (133). Similar re-sults were obtained in the study of a transmem-brane fragment of human CD4, in which thesignals observed were thought to originate fromflexible, solvent-exposed regions (134). Muchmore complete data were obtained recently us-ing perdeuterated VDAC-1 (135). The rota-tional correlation time, τ c, of VDAC-1 in NDswas estimated to be ∼93 ns, only slightly longerthat the value of ∼70 ns observed in LDAOmicelles. As larger and larger MPs are beingstudied, the extra bulk added by the MSPs andlipids ought to become less and less of a handi-cap. One may also note that, whereas the slowtumbling rate of MP/ND complexes compli-cates structural studies of the protein itself, itwould not prevent, for instance, studying the


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structure of bound ligands by transferred NOEmeasurements.

ssNMR is well adapted to the study of largeobjects. It has been used, as mentioned above(Section 3.1), to study the structure of MSPs inNDs that had been precipitated using polyethy-lene glycol (50). Upon precipitation of NDscontaining the human P450 cytochrome 3A4(CYP3A4), the protein retained its ability tobind one of its ligands, bromocryptine. 13Cchemical shifts were consistent with the knownX-ray structure of CYP3A4, illustrating the fea-sibility of studying by ssNMR the structure ofND-embedded MPs (136).

ssNMR of MP/APol complexes has not beenattempted yet. It should be feasible, given thatthe complexes can be precipitated or frozen and,at least in some cases, lyophilized without de-naturing the protein (Section 5.1). However, agreat advantage of ssNMR is that it gives ac-cess to the protein in its natural environment,which is maintained in lipid bilayers and, to alarge extent, in NDs and in bicelles, but notin APols. APols could, however, be useful, e.g.,to study the structure of bound ligands or ofsmall isotopically labeled subunits integratedinto large unlabeled complexes, which currentNDs would not be able to trap.

7.6. Crystallization

A8-35, the best-characterized APol, is not a pri-ori an excellent candidate for forming 3D crys-tals of MP/APol complexes because of the highdensity of charges it carries (no structure of MPhas ever been solved using a charged detergent).Indeed, attempts at crystallizing cytochromebc1/A8-35 complexes did not yield any crys-tals (D. Charvolin, unpublished data). Somepoorly diffracting crystals were obtained in pre-liminary studies of ternary bc1/A8-35/detergentcomplexes (D. Charvolin, A.-N. Galatanu, &M. Picard, unpublished data). The color andspace group of these crystals, as well as theirfluorescence when they were prepared usinga fluorescent APol, indicated that they con-tained the protein, the polymer, and, not showndirectly but necessarily (see Section 5.1), the

detergent. It has not yet been possible, unfor-tunately, to determine whether the poor qual-ity of the diffraction patterns observed (∼20-Aresolution) is inherent to this type of crystals orresults from their handling, from the freezingstep, or, simply, from too few crystals havingbeen examined. Why crystals were obtained internary mixtures and not with pure APols canhave several origins. One is that diluting A8-35with detergent will diminish the charge densityof the surfactant belt surrounding the proteinas well as facilitate charge redistribution in thecrystal. Another may be a more homogeneoussize of the complexes, as suggested by obser-vations on tOmpA/A8-35 versus tOmpA/A8-35/C8E4 complexes (93) (Section 5.1). Crys-tallization attempts with NAPols may be ex-pected to have greater chances of success. Forthe time being, APols cannot be advocated as afavorable medium for MP crystallization. How-ever, because even mixtures of APols and de-tergent can have a stabilizing effect on MPs(Section 6.1), supplementing an MP in deter-gent solution with APols, used as an additive,may perhaps be attempted when protein insta-bility is suspected to be the reason for the failureto grow good-quality crystals in pure detergentsolution.

Attempts to grow 3D crystals from solutionsof MPs in (H)FSs should preferably be carriedout using chemically defined molecules, whichhave become available only very recently (71,72). One may speculate that, because their affin-ity for MP transmembrane surfaces is likely tobe relatively low, (H)FSs may favor the growthof type I crystals, which are made of stacks of2D crystals.

Detergents do not displace or dissolvemonolayers of (H)FSs, which are stronger sur-factants and do not mix well with them. Nickel-bearing (H)FSs have been synthesized (137,138). They can be used, as nickel-bearing flu-orinated lipids have been in the past (139),to adsorb detergent-solubilized polyhistidine-tagged MPs at the air-water interface. Thus,His-tagged tOmpA/C8E4 complexes can dif-fuse within the aqueous space separating thetwo F-TAC monolayers of a Newton black

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film, where they form, depending on the condi-tions, one- or two-molecule thick layers, whosestructure has been studied by X-ray reflectivitymeasurements (137).

HFS monolayers containing Phenyl-HF-NTANi (Figure 3c) may hold interestingpromises for forming 2D MP crystals (138). Todate, HFS monolayers have been shown to al-low adsorption and orientation of the sulfony-lurea receptor, whose projection structure wasthen solved by single-particle EM imaging (C.Venien-Bryan, unpublished data).

Solving MP structures by X-ray analysis of3D crystals of MP/ND complexes would de-pend, in most cases, on the target MP lockingitself in perfect register with the MSPs, which,although it could conceivably be engineered,may be a rare circumstance.

7.7. Single-Particle ElectronMicroscopy and AtomicForce Microscopy

As described in Section 3.1, empty NDs havebeen studied by EM and AFM. NDs can be usedfor single-particle analyses of embedded MPs,with the limitation that they will not be able totrap MPs with very large transmembrane do-mains. This is a handicap for EM single-particlestudies, which work best on large objects. Uponexposure to a mica surface, NDs, whether ornot they harbor an MP, will tend to lie flat onthe surface, which greatly facilitates AFM stud-ies of their structure and that of the MP. AFM,for instance, has been used to measure the ex-tension of CYP2B4 and the cytochrome P450reductase in a direction normal to the plane ofthe disc (Reference 41 and references therein).Single-particle EM imaging, coupled with theuse of antibody Fab fragments, has shown that,in NDs containing two rhodopsin molecules,the two proteins adopt indifferently parallel orantiparallel orientations with respect to eachother (44).

APols and (H)FSs have the potential to trapand stabilize very large MP complexes, whichmay facilitate their purification, the identifica-tion of their components, and the study of their

structure by single particle EM and other ap-proaches. This application, which seems bothpromising and relatively straightforward, hasbeen underexploited. In an early study, scan-ning transmission EM, which is mostly use-ful to map the distribution of masses ratherthan for imaging, had been used to show thattrapping with A8-35 captured the distributionof cytochrome b6f dimers and monomers thatpreexisted in the original detergent solution(140). To date, four single-particle EM stud-ies have been published, two studies of the A8-75-trapped proton ATP synthase (141, 142), anegative-stain study of A8-35-trapped BR andof the curious filaments these complexes formupon elimination of free APol (83), and a low-resolution cryo-EM study of A8-35-trappedmitochondrial complex I (143). As already men-tioned, A8–35 has been used to trap and purifyunder a functional form mitochondrial super-complex B, which comprises complexes I, III,and IV in a 1:2:1 molar ratio. The complexeswere imaged both after negative staining and bycryo-EM (Figure 9); image reconstruction is inprogress (T. Althoff & W. Kuhlbrandt, personalcommunication).

No EM studies of MPs stabilized by (H)FSshave been reported yet.

7.8. Delivering Membrane Proteinsto Preformed Lipid Bilayers

As mentioned above, APols and (H)FSs, undermost conditions, will not dissolve lipid mem-branes, and they are not cytolytic (Sections 5.1and 5.3). They can, therefore, be used to deliverMPs to lipid vesicles, planar bilayers, or cellswithout destroying them. This useful propertyhas permitted integration of APol-trapped dia-cylglycerol kinase into lipid vesicles (80), of A8-35-trapped porins into lipid black films (123),of the mechanosensitive channel MscL, keptsoluble by (H)F-TAC, into lipid vesicles (59),and of APol-trapped mimics of transmembranepeptides into living cells (G. Cremel, personalcommunication). In such studies, however, twocaveats have to be kept in mind: (a) fragile MPsrisk being denatured in the process (BR cannot


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a b

50 nm 50 nm

Figure 9Electron microscopy of amphipol-trapped mitochondrial supercomplex B. The supercomplex (∼1.7 MDa),which comprises one copy each of complexes I and IV and two copies of complex III, was trapped by APolA8-35, purified, and imaged either by negative staining (a) or in vitrous ice (b) (T. Althoff & W. Kuhlbrandt,in preparation).

be directly transferred from (H)FSs to lipidvesicles without denaturation) (C. Breyton,unpublished observation); and (b) whereas(H)FSs can be washed away, as they will equili-brate between the solution and the membrane,APols will remain associated with the targetmembrane, which may have undesirable con-sequences (see Reference 123).

The unusual situation of being able to keepa hydrophobic protein soluble, thanks to a sur-factant, without lysing the target membranehas been exploited in a series of studies of thethermodynamics of insertion of the T domainof diphteria toxin into membranes following apH drop (Figure 10) (58, 61, 144). Protein/surfactant interactions were examined byFRET thanks to Oregon Green–labeled (H)F-TACs (Figure 3d ) (61).

Transfer of an MP from an MP/ND com-plex to a preformed membrane, an intriguingpossibility, has not been reported yet. The ab-sence of transfer, however, would make it fea-sible to study the interaction of a membrane-embedded MP with an ND-trapped one, orof two ND-trapped MPs with one another,while the two proteins are kept in distinct bi-layers. This would provide a novel way to study

interactions between MPs that in vivo belongto distinct membranes, as occurs, for instance,in processes involving membrane adhesion orfusion.

7.9. Immobilizing Membrane Proteinsonto Solid Supports

Surface-based in vitro assays of ligand bindingto proteins present multiple advantages, espe-cially their adaptability to the use of minimalamounts of proteins and reagents and tomultiplexing and high-throughput screening.Immobilizing an MP without modifying it orsubjecting it to nonspecific, potentially dena-turing interactions is, however, a challenge.Because the association of APols with MPsresists extensive washing with surfactant-freebuffer (see Section 5.1), trapping an MPwith a functionalized APol results in thepermanent association with the protein ofany functional group carried by the APol(93). Tagged APols thus can be used to im-mobilize MPs onto solid supports (92). Theligand-binding properties of the immobilizedproteins can then be studied in detergent-free solutions, e.g., by SPR or fluorescence

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pH 7.0 pH 4.5

HF-TAC

I II IIa

IIIIV

Interfacial intermediate

T domain

Membrane

Pathway 1

Pathway 2

Insertedstate

Figure 10Using (H)FSs to study the thermodynamics of protein insertion into membranes. The pore-forming Tdomain of diphteria toxin, which is water soluble at pH 7.0 (I), changes conformation as the consequence ofa pH drop, which renders it hydrophobic (II). In the absence of surfactants, a competition engages betweenmembrane insertion (III, pathway 1) and nonproductive aggregation (IIa). HF-TAC keeps the T domainsoluble and monomeric without solubilizing the target membrane, at variance with what a conventionaldetergent would do. This makes it possible to study the thermodynamics of insertion under equilibriumconditions (see Section 7.8). From Reference 58.

techniques, opening the way to applications indiagnostics, drug discovery, or the search fornatural biological partners (Figure 11). Theprocedure is universal in the sense that anyMP is a priori amenable to it. It is also mild,because the protein itself is not involved inany interaction with the support, and highlyversatile.

NDs can adsorb onto solid supports, eitherspontaneously or via tagged lipids, which can beused to create patterned bilayer surfaces (145,146). His-tagged scaffolding proteins wereused to form NDs incorporating rhodopsin,which were immobilized onto a nickel-bearingsupport, and SPR and a modified versionof MALDI-TOF mass spectrometry wereused to demonstrate transducin binding uponexposure to light (147). Similarly, His-taggedNDs were used to capture glycolipid GM1,the membrane receptor for cholera toxin.After immobilization of the complexes ontoNi-NTA chips, the kinetics of the interactionof the soluble toxin with the immobilized lipid

was studied by SPR (148). Recent studies havedescribed the screening of ligands of immobi-lized ND-trapped MPs by SPR (149) or NMR(150).

7.10. Folding Membrane Proteinsfrom a Denatured State

Folding MPs to their native state in vitroprovides precious insights into the respectiveroles of the amino acid sequence, the inser-tion machinery, and the membrane environ-ment in determining the 3D fold adopted bythe polypeptide. From a practical point ofview, overexpressing MPs as inclusion bod-ies and folding them in vitro may allow pro-duction of large amounts of naturally rareMPs, which is often difficult to achieve whentrying to express them directly in a func-tional state. Mild surfactants, such as APolsand (H)FSs, may a priori provide favor-able media for MPs to fold because theyare expected to interfere less than detergents


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150

100

Anti-tOmpA Anti-BR Anti-b6f Anti-bc1

Antibodies flushed over chip

Time (s)

Re

spo

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(R

U) Material adsorbed

onto channel:

BAPol

tOmpA/BAPol

BR/BAPol

b6f/BAPol

bc1/BAPol

Membraneprotein

Functionalizedamphipol

Complementarysurface

Antibody

LigandDetergent

1 2 3 4

50

0

0 500 1000 1500 2000 2500–50

Pre-immune Preimmune m

Figure 11Using functionalized amphipols to attach membrane proteins onto solid supports and detect ligand bindingto them. (top) Principle. A membrane protein (MP) in detergent solution ©1 is trapped with a biotinylatedamphipol (BAPol) ©2 . It is then attached via the biotin groups to a streptavidin-coated chip ©3 . Ligands areflushed over it in plain, surfactant-free buffers, and their binding is detected by any of various methods ©4 .(bottom) Validation. Channels of a streptavidin-coated chip were exposed either to pure BAPol or tocomplexes of BAPol with any of four different MPs: tOmpA, BR, and cytochromes b6 f and bc1. The fivechannels were then flushed with buffer containing either preimmune antibodies or antibodies raised againsteach of the four MPs. Antibody binding, measured in response units (RUs) by surface plasmon resonance, isprotein specific. Adapted from Reference 92.

with the protein/protein and protein/lipidinteractions that determine the native 3D struc-ture of MPs and that stabilize them. APol A8-35 indeed has proven to be a highly efficientmedium for folding BR, two porins, and severalGPCRs (Figure 12) (120, 123). Generalizationof this strategy could constitute a major break-through for structural and functional studies ofmany MPs. From a fundamental point of view,these experiments show that all of the chemi-cal information needed for these MPs to cor-rectly fold is stored in their sequences and thatthe highly specific and anisotropic environmentprovided by a lipid bilayer is not required to de-code this information.

The use of (H)FSs to fold MPs has notbeen extensively investigated yet. Preliminarytests show that BR can be refolded with morethan 50% yield in the presence of either F- orHF-TAC (60). tOmpA could be refolded into(H)FSs as well, albeit to a lesser extent thanwhen using conventional detergents; HF-TACwas more efficient than F-TAC (60). The use-fulness of (H)FSs for MP folding clearly de-serves further investigation.

Refolding MPs into NDs is a more complexproposition because it will generally call for si-multaneous refolding of the target MP and thescaffolding proteins as well as their reassemblywith the lipids. Nevertheless, it may be worth

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BLT2 CB15-HT4(a)

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01:5 1:10 1:20 1:5 D+L A A+L D+L A A+L D+L A A+L

Figure 12Amphipol-assisted folding of four G protein–coupled receptors (GPCRs) expressed in an inactive form ininclusion bodies. The BLT1 leukotriene receptor (left) and the 5-HT4(a) serotonin receptor, the BLT2leukotriene receptor, and the CB1 cannabinoid receptor (right), all of them class A GPCRs, were expressedin inclusion bodies and purified in an inactive form in sodium dodecyl sulfate (SDS) solution. They werefolded by substituting SDS either with a lipid/detergent mixture (D+L), with pure A8-35 (A) at differentprotein/APol mass ratios (BLT1), with A8-35 (A) at a 1:5 mass ratio (BLT2, 5-HT4(a) and CB1), or withA8-35 supplemented with asolectin in a 1:5:1 protein/APol/asolectin mass ratio (A+L). The extent of correctfolding is expressed as the percentage of total receptor (on the basis of the protein concentration in the SDSsolution) that is able to bind a specific ligand. Adapted from Reference 120.

examining whether NDs would not reform, forinstance, from a mixture of their constituents inSDS solution, in which case MP folding exper-iments might be attempted.

7.11. Expressing Membrane Proteinsin Cell-Free Systems

Cell-free expression of MPs in a lysatecontaining the transcription and translationmachineries offers highly interesting opportu-nities, e.g., for expressing MPs that are toxic tocells as well as for specific labeling. Tradition-ally, MPs are expressed in vitro in the presenceof a detergent, of lipid vesicles, or in the absenceof any surfactant at all. In the latter case, MPsprecipitate and are later solubilized using a de-tergent. Detergents have the drawback of beinginactivating, and lipids have the disadvantageof offering limited yields. There is probably anadvantage in the use of NCSs as less-aggressiveenvironments, in which neosynthesized MPsshould stand a better chance to correctly fold.All three systems can be used to this end.(H)F-TACs have been used to synthesize themechanosensitive channel MscL (59). Aftersynthesis, MscL was purified in solutions of the

same surfactants and directly applied to lipo-somes, into which it spontaneously integrated.Its activity was then characterized by patch-clamp measurements (59). MscL synthesis hasalso been achieved using the new, chemicallydefined (H)FSs (71). NDs have been employedto synthesize under their functional form BRand the multidrug-resistance protein EmrE, aswell as to express a large panel of other MPs(151–153). APols initially yielded disappoint-ing results. Indeed, both A8-35 and sulfonatedAPols were found to inhibit the transcription-translation machinery (E. Billon-Denis &F. Zito, unpublished observations). Morerecently, however, it was observed that NAPolsdo not block protein synthesis and provideexcellent yields of expression for several MPs(89; E. Billon-Denis, P. Bazzacco, & F. Zito,unpublished observations). BR expressed inthe presence of NAPols is both functional andsoluble.

These observations are potentially of greatinterest. Given that most MPs are more or lessunstable in detergent solutions and that foldingyields in detergent or detergent/lipid mixturesare generally low, it is to be expected that many,if not most, MPs, if exposed to detergents in


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Table 1 Opportunities and constraints associated with the use of amphipols, nanodiscs, and fluorinated surfactantsa,b

Technology Amphipols NanodiscscFluorinatedsurfactants Sectionsd

Membrane protein (MP) stabilization + + + 6Functional studies + ++ + 7.3Mediating MP immobilization for ligand binding measurements + + − 7.9Optical spectroscopy (visible absorption spectrum) + + + 7.1Optical spectroscopy (UV, intrinsic MP fluorescence, circulardichroism)

+ ± + 7.1

Fluorescence spectroscopy using probes + + + 7.1Infrared spectroscopy − ± − 7.1MP solution studies by AUC, SEC, SAXS, SANS + ± + 7.2Solution NMR + ±? ? 7.5Solid-state NMR +? + − 7.5Three-dimensional crystallization ± −? +? 7.6Two-dimensional crystallization − −? +? 7.6Trapping MP supercomplexes + ± + 5, 7.7EM, AFM (single particles) + + + 7.7Transferring MPs to preformed membranes + ? + 7.8Folding full-length MPs to native state + ? + 7.10MP cell-free translation + + + 7.11Isoelectrofocusing with two-dimensional gels + +? ? 7.4MP mass spectrometry + ? ? 7.4

a+, ± , and – signs refer to how promising or problematical each application to MP studies looks: +, promising; ± , promising, but with limitations ordifficulties; −, not promising if not plainly impossible.bFor those applications that have actually been tested, cells have been colored as follows: green, tested with success; yellow, shown to work, but with somecaveats or limitations; pink, problematical if not impossible. A white background indicates that the +/- signs represent what appears to be reasonableexpectations, but that these assessments are not currently backed up by actual data.cIn the case of nanodiscs, the table refers to the study of nanodisc-embedded MPs, not to that of rim proteins.dSection of this article where this is discussed.Abbreviations: AFM, atomic force microscopy; AUC, analytical ultracentrifugation; EM, electron microscopy; SANS, small-angle neutron scattering;SAXS, small-angle X-ray scattering; SEC, size exclusion chromatography; UV, ultraviolet.

the course of their synthesis, will not efficientlyachieve their native 3D fold. NCSs may there-fore offer an extremely attractive alternative forcell-free MP synthesis.

8. CONCLUSION:OPPORTUNITIES VERSUSCONSTRAINTS

It is my hope that the present article, which, un-fortunately, had to limit itself to three amongmany more innovative systems, will convincemembrane protein biochemists and biophysi-cists that the time is over when MPs could be

handled in vitro only in a detergent solution orin a membrane-bound form. New tools havebeen developed and validated—painstakingly!These tools can at least partially circumventthe instability and other problems encounteredwith detergents. They can also provide totallynew experimental opportunities. In Table 1, Ihave summarized my personal view of the use-fulness of the three NCS systems discussed hereas well as the constraints and difficulties withwhich each is associated. Needless to say, theseprospects will evolve as improved moleculesare developed and applications are more thor-oughly explored.

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As should be evident from the present re-view, each type of NCS has its own privi-leged applications. Although some uses, suchas MP folding or in vitro synthesis, can prob-ably be advantageously developed in paral-lel with the three of them, specialization will

occur. One may speculate that, ten yearsfrom now, membrane biochemists and bio-physicists may be in the habit of resort-ing to one or the other NCS dependingon which particular problem they have athand.

SUMMARY POINTS

1. Membrane proteins (MPs) can be handled in detergent-free aqueous solutions as individ-ual, well-defined complexes after being transferred to nondetergent surfactants, includingamphipols (APols), nanodiscs (NDs), or fluorinated surfactants (FSs).

2. Transfer is usually achieved by first solubilizing the target protein with a detergent andthen replacing the detergent with any of these three media.

3. Transferring MPs to any of these three environments generally stabilizes them as com-pared to detergent solutions.

4. Some applications, such as cell-free synthesis of MPs, the study of fragile MP complexes,or single-particle imaging, have been shown or appear likely to be common to all threesystems. Others are best performed using one or the other of them. The development ofmany applications, however, is still in its infancy.

5. NDs have the unique characteristic of featuring a well-defined area into which onlyMPs whose transmembrane domain does not exceed a defined size can be trapped in amembrane-like environment. This makes them exceptional tools to sort out functionalissues related to MP oligomerization and interactions with lipids. Other applicationswhose development has started include electron microscopy, atomic force microscopy,solution and solid-state NMR, and MP immobilization.

6. Among applications for which APols appear particularly promising are MP folding, so-lution NMR, the study of large MP complexes, and immobilization of MPs onto solidsupports.

7. Applications of FSs are at a less developed stage, but among those that may be partic-ularly useful are the study of fragile complexes and the delivery of MPs to preexistingmembranes.

8. All three systems have by now been studied extensively enough to be used for the explo-ration of biological systems whenever detergents are unsuitable.

FUTURE ISSUES

1. One can expect that future developments will take, for each of the three systems that havebeen discussed here, two main paths: improving the tools and exploring applications.

2. Regarding the development of applications, several suggestions have been evoked inSection 7 of the review. Exploring higher and less-permanent levels of organization thanthat of the single macromolecule is one of the frontiers of today’s biology. Means topreserve fragile or transient interactions between MPs better than is possible using de-tergents, which nonconventional surfactants provide, are, therefore, particularly timely.


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3. Regarding the development of the tools, the resources of genetic engineering will improveand diversify the proteins used to stabilize NDs, which will permit them to encapsulatelarger MPs or to more precisely fix the size of the discs, to tailor their spectroscopicproperties to the needs of biophysicists, or to functionalize them.

4. Functionalization is also a key to developing original applications of APols. The new APolgenerations, and in particular nonionic APols, will have to be as thoroughly validated asfirst-generation molecules have been, and optimized and functionalized in view of theirspecific uses.

5. Whether developing chemically well-defined APols—a difficult challenge to chemists—is worth the long effort required by such an attempt remains to date an open question.Developing and validating applications is probably more urgent.

6. Fluorinated surfactants have been brought to the stage where chemically definedmolecules are, at long last, available. Much effort is still necessary, however, to morefully explore their specific applications.

7. Finally, one should not forget that the three systems analyzed here do not cover the wholerange of approaches that have been explored to date and that novel ones will no doubtemerge.

DISCLOSURE STATEMENT

The author is coinventor on several granted or pending patents on amphipols and their uses.

ACKNOWLEDGMENTS

Particular thanks are due to T. Althoff, J.-L. Baneres, S. Banerjee, P. Bazzacco, C. Bechara, E.Billon-Denis, G. Bolbach, J. Borch, C. Breyton, L. Catoire, P. Champeil, D. Charvolin, G. Cremel,T. Dahmane, F. Giusti, Y. Gohon, E. Goormaghtigh, J.-C. Guillemot, T. Hamann, A. Krisko, W.Kuhlbrandt, A. Ladokhin, C. Le Bon, M. le Maire, K.L. Martinez, A. Milon, B.L. Møller, E.Pebay-Peyroula, M. Picard, Y. Pierre, R. Prassl, B. Pucci, M. Renault, C.M. Rienstra, J.N. Sachs,S. Sagan, T.P. Sakmar, C.R. Sanders, S.G. Sligar, R. Sunahara, C.G. Tate, C. Tribet, C. Venien-Bryan, F. Wien, F. Winnik, F. Zito, and M. Zoonens for communication and permission to quoteunpublished information and/or to reproduce published or unpublished figures, for help with thefigures or the bibliography, and for comments on the manuscript. I am deeply indebted to J. Barrafor her help with preparing the figures and with collecting the bibliography and to M.E. Dumontfor his heroic attempts at improving the English wording. My own work has been mainly supportedby the CNRS, the Human Frontier Science Program Organization (RG00223/2000-M), and theEuropean Community (BIO4-CT98-0269 and STREP LSHG-CT-2005-513770 Innovative Toolsfor Membrane Protein Structural Proteomics).

This review is dedicated to the memory of my father, whose courage and thoroughness havebeen an inspiration for me.

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108. Catoire LJ, Zoonens M, van Heijenoort C, Giusti F, Guittet E, Popot J-L. 2009. Solution NMR mappingof water-accessible residues in the transmembrane β-barrel of OmpX. Eur. Biophys. J. PMID:19639312.In press

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110. Bayburt TH, Sligar SG. 2003. Self-assembly of single integral membrane proteins into soluble nanoscalephospholipid bilayers. Protein Sci. 12:2476–81

111. Bayburt TH, Grinkova YV, Sligar SG. 2006. Assembly of single bacteriorhodopsin trimers in bilayernanodiscs. Arch. Biochem. Biophys. 450:215–22

112. Boldog T, Grimme S, Li M, Sligar SG, Hazelbauer GL. 2006. Nanodiscs separate chemoreceptoroligomeric states and reveal their signaling properties. Proc. Natl. Acad. Sci. USA 103:11509–14

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114. Leitz AJ, Bayburt TH, Barnakov AN, Springer BA, Sligar SG. 2006. Functional reconstitution of β2-adrenergic receptors utilizing self-assembling nanodisc technology. Biotechniques 40:601–12

115. Whorton MR, Bokoch MP, Rasmussen SG, Huang B, Zare RN, et al. 2007. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein. Proc.Natl. Acad. Sci. USA 104:7682–87

116. Yao XJ, Velez Ruiz G, Whorton MR, Rasmussen SG, DeVree BT, et al. 2009. The effect of ligand efficacyon the formation and stability of a GPCR-G protein complex. Proc. Natl. Acad. Sci. USA 106:9501–6

117. Bayburt TH, Leitz AJ, Xie G, Oprian DD, Sligar SG. 2007. Transducin activation by nanoscale lipidbilayers containing one and two rhodopsins. J. Biol. Chem. 282:14875–81


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119. Shih AY, Denisov IG, Phillips JC, Sligar SG, Schulten K. 2005. Molecular dynamics simulations ofdiscoidal bilayers assembled from truncated human lipoproteins. Biophys. J. 88:548–56

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121. Nehme R, Joubert O, Bidet M, Lacombe B, Polidori A, et al. 2010. Stability study of the human Gprotein–coupled receptor, Smoothened. Biochim. Biophys. Acta. In press

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124. Duarte AMS, Wolfs CJAM, Koehorsta RBM, Popot J-L, Hemminga MA. 2008. Solubilization of V-ATPase transmembrane peptides by amphipol A8-35. J. Peptide Chem. 14:389–93

125. Nelson RG, Johnson WC Jr. 1972. Optical properties of sugars. I. Circular dichroism of monomers atequilibrium. J. Am. Chem. Soc. 94:3343–45

126. Baas BJ, Denisov IG, Sligar SG. 2004. Homotropic cooperativity of monomeric cytochrome P450 3A4in a nanoscale native bilayer environment. Arch. Biochem. Biophys. 430:218–28

127. Joubert O, Nehme R, Fleury D, de Rivoyre M, Bidet M, et al. 2009. Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast. Biochim. Biophys. Acta1788:1813–21

128. Whorton MR, Jastrzebska B, Park PS, Fotiadis D, Engel A, et al. 2008. Efficient coupling of transducinto monomeric rhodopsin in a phospholipid bilayer. J. Biol. Chem. 283:4387–94

129. Shaw AW, Pureza VS, Sligar SG, Morrissey JH. 2007. The local phospholipid environment modulatesthe activation of blood clotting. J. Biol. Chem. 282:6556–63

130. Morrissey JH, Pureza V, Davis-Harrison RL, Sligar SG, Ohkubo YZ, Tajkhorshid E. 2008. Blood clottingreactions on nanoscale phospholipid bilayers. Thromb. Res. 122(Suppl. 1):S23–66

131. Rabilloud T. 2009. Membrane proteins and proteomics: love is possible, but so difficult. Electrophoresis30:S174–80

132. Gohon Y. 2002. Etude structurale et fonctionnelle de deux proteines membranaires, la bacteriorhodopsine et lerecepteur nicotinique de l’acetylcholine, maintenues en solution aqueuse non detergente par des polymeres am-phiphiles. PhD thesis. Univ. Paris-VI, Paris. 467 pp.

133. Lyukmanova EN, Shenkarev ZO, Paramonov AS, Sobol AG, Ovchinnikova TV, et al. 2008. Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins andmembrane-active peptides. J. Am. Chem. Soc. 130:2140–41

134. Gluck JM, Wittlich M, Feuerstein S, Hoffmann S, Willbold D, Koenig BW. 2009. Integral membraneproteins in nanodiscs can be studied by solution NMR spectroscopy. J. Am. Chem. Soc. 131:12060–61

135. Raschle T, Hiller S, Yu TY, Rice AJ, Walz T, Wagner G. 2009. Structural and functional characterizationof the integral membrane protein VDAC-1 in lipid bilayer nanodiscs. J. Am. Chem. Soc. 131:17777–79

136. Kijac AZ, Li Y, Sligar SG, Rienstra CM. 2007. Magic-angle spinning solid-state NMR spectroscopy ofnanodisc-embedded human CYP3A4. Biochemistry 46:13696–703

137. Petkova V, Benattar J-J, Zoonens M, Zito F, Popot J-L, et al. 2007. Free-standing films of fluorinatedsurfactants as 2D matrices for organizing detergent-solubilized membrane proteins. Langmuir 23:4303–9

138. Dauvergne J, Polidori A, Venien-Bryan C, Pucci B. 2008. Synthesis of a hemifluorinated amphiphiledesigned for self-assembly and two-dimensional crystallization of membrane proteins. Tetrahedron Lett.49:2247–50

139. Lebeau L, Lach F, Venien-Bryan C, Renault A, Dietrich J, et al. 2001. Two-dimensional crystallizationof a membrane protein on a detergent-resistant lipid monolayer. J. Mol. Biol. 308:639–47

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Escherichia coli F1F0-ATPsynthase by immuno-electron microscopy: The δ subunit binds on top of theF1. J. Mol. Biol. 295:387–91

143. Flotenmeyer M, Weiss H, Tribet C, Popot J-L, Leonard K. 2007. The use of amphipathic polymers forcryo-electron microscopy of NADH:ubiquinone oxidoreductase (complex I). J. Microsc. 227:229–35

144. Posokhov YO, Rodnin MV, Das SK, Pucci B, Ladokhin AS. 2008. FCS study of the thermodynamicsof membrane protein insertion into the lipid bilayer chaperoned by fluorinated surfactants. Biophys. J.95:L54–56

145. Goluch ED, Shaw AW, Sligar SG, Liu C. 2008. Microfluidic patterning of nanodisc lipid bilayers andmultiplexed analysis of protein interaction. Lab Chip 8:1723–28

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148. Borch J, Torta F, Sligar SG, Roepstorff P. 2008. Nanodiscs for immobilization of lipid bilayers andmembrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor. Anal. Chem.80:6245–52

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Annual Review ofBiochemistry

Volume 79, 2010Contents

Preface

The Power of OneJames E. Rothman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �v

Prefatory Article

FrontispieceAaron Klug � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � xiv

From Virus Structure to Chromatin: X-ray Diffractionto Three-Dimensional Electron MicroscopyAaron Klug � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Recent Advances in Biochemistry

Genomic Screening with RNAi: Results and ChallengesStephanie Mohr, Chris Bakal, and Norbert Perrimon � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �37

Nanomaterials Based on DNANadrian C. Seeman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �65

Eukaryotic Chromosome DNA Replication: Where, When, and How?Hisao Masai, Seiji Matsumoto, Zhiying You, Naoko Yoshizawa-Sugata,

and Masako Oda � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �89

Regulators of the Cohesin NetworkBo Xiong and Jennifer L. Gerton � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 131

Reversal of Histone Methylation: Biochemical and MolecularMechanisms of Histone DemethylasesNima Mosammaparast and Yang Shi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 155

The Mechanism of Double-Strand DNA Break Repair by theNonhomologous DNA End-Joining PathwayMichael R. Lieber � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 181

The Discovery of Zinc Fingers and Their Applications in GeneRegulation and Genome ManipulationAaron Klug � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 213

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Origins of Specificity in Protein-DNA RecognitionRemo Rohs, Xiangshu Jin, Sean M. West, Rohit Joshi, Barry Honig,

and Richard S. Mann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 233

Transcript Elongation by RNA Polymerase IILuke A. Selth, Stefan Sigurdsson, and Jesper Q. Svejstrup � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 271

Biochemical Principles of Small RNA PathwaysQinghua Liu and Zain Paroo � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 295

Functions and Regulation of RNA Editing by ADAR DeaminasesKazuko Nishikura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 321

Regulation of mRNA Translation and Stability by microRNAsMarc Robert Fabian, Nahum Sonenberg, and Witold Filipowicz � � � � � � � � � � � � � � � � � � � � � � � � 351

Structure and Dynamics of a Processive Brownian Motor:The Translating RibosomeJoachim Frank and Ruben L. Gonzalez, Jr. � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 381

Adding New Chemistries to the Genetic CodeChang C. Liu and Peter G. Schultz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 413

Bacterial Nitric Oxide SynthasesBrian R. Crane, Jawahar Sudhamsu, and Bhumit A. Patel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 445

Enzyme Promiscuity: A Mechanistic and Evolutionary PerspectiveOlga Khersonsky and Dan S. Tawfik � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 471

Hydrogenases from Methanogenic Archaea, Nickel, a Novel Cofactor,and H2 StorageRudolf K. Thauer, Anne-Kristin Kaster, Meike Goenrich, Michael Schick,

Takeshi Hiromoto, and Seigo Shima � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 507

Copper MetallochaperonesNigel J. Robinson and Dennis R. Winge � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 537

High-Throughput Metabolic Engineering: Advances inSmall-Molecule Screening and SelectionJeffrey A. Dietrich, Adrienne E. McKee, and Jay D. Keasling � � � � � � � � � � � � � � � � � � � � � � � � � � 563

Botulinum Neurotoxin: A Marvel of Protein DesignMauricio Montal � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 591

Chemical Approaches to GlycobiologyLaura L. Kiessling and Rebecca A. Splain � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 619

Cellulosomes: Highly Efficient Nanomachines Designed toDeconstruct Plant Cell Wall Complex CarbohydratesCarlos M.G.A. Fontes and Harry J. Gilbert � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 655

viii Contents

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Somatic Mitochondrial DNA Mutations in Mammalian AgingNils-Goran Larsson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 683

Physical Mechanisms of Signal Integration by WASP Family ProteinsShae B. Padrick and Michael K. Rosen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 707

Amphipols, Nanodiscs, and Fluorinated Surfactants: ThreeNonconventional Approaches to Studying Membrane Proteins inAqueous SolutionsJean-Luc Popot � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 737

Protein Sorting Receptors in the Early Secretory PathwayJulia Dancourt and Charles Barlowe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 777

Virus Entry by EndocytosisJason Mercer, Mario Schelhaas, and Ari Helenius � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 803

Indexes

Cumulative Index of Contributing Authors, Volumes 75–79 � � � � � � � � � � � � � � � � � � � � � � � � � � � 835

Cumulative Index of Chapter Titles, Volumes 75–79 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 839

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org

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