BIOC. 585: BIOLOGICAL MEMBRANES

!             Overview  biological roles  structural features

!             Membrane lipids  general structures   aggregation states; polymorphism  thermal transitions   electrostatic effects   molecular dynamics (translational and rotational diffusion,

flip-flop) 

!             Membrane proteins  crystallization  overview of structural features  structure/function relations:

photosynthetic electron transfer (cyt b6f)ion transport (K+ channels)bacteriorhodopsin

READING LIST

Biochem. 585: Membrane Proteins Reading List [all articles in pdf files on course website]MEMBRANE PROTEIN CRYSTALLIZATION

Ostermeier & Michel, “Crystallization of membrane proteins”, Curr. Op. Struct. Biol. 7, 697-701 (1997). Hunte & Michel, “Crystallization of membrane proteins mediated by antibody fragments”, Curr. Op.Struct.

Biol. 12, 503-508 (2002).STRUCTURES AND FUNCTIONS

OverviewScarlata, "Membrane Protein Structure", Chap. 1, Section 2, Biophysical Soc. on-line textbook.Byrne & Iwata, “Membrane protein complexes”, Curr. Opinion in Struct. Biol. 12, 239-243 (2002).Shipley, "Lipids; Bilayers and non-bilayers: structures, forces and protein crystallization", Curr. Op. Struct.

Biol. 10, 471-473 (2000). Electron Transfer in Photosynthesis

Optional: Golbeck, “Photosynthetic Reaction Centers”, Biophysical Soc. on-line textbook.Kurisu et al., “Structure of the cytochrome b6f complex of oxygenic photosynthesis” Science 302, 1009-

1014 (2003).Stroebel et al., “An atypical heme in the cytochrome b6f complex”, Nature 426, 413-418 (2003).Khlbrandt “Dual approach to a light problem”, Nature 426, 399-400 (2003)

Ion ChannelsDoyle et al. “The structure of the potassium channel: molecular basis of K+ conduction and selectivity”,

Science 280, 69-77 (1998).Rees et al. “Crystallographic analysis of ion channels: lessons and challenges” J. Biol. Chem. 275, 713-

716 (2000).MacKinnon “Potassium channels” FEBS Letters 555, 62-65 (2003).Jiang et al. “X-ray structure of a voltage-dependent K+ channel”, Nature 423, 33-41 (2003).

BacteriorhodopsinLanyi “Bacteriorhodopsin”, Bioenergetics, Chap. 3, Biophysical Soc. on-line textbook.Optional: Neutze et al. “Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport”

Biochim. Biophys. Acta 1565, 144-167 (2002).

BIOLOGICAL ROLES OF MEMBRANES

selective permeability barriers (cell compartmentalization): pumps, gates, sieves

structural organization of cellular processes (e.g. energy transduction): respiration, photosynthesis, vision

receptors for external stimuli: hormones, neurotransmitters

cell recognition: immune response, tissue formation

intercellular communication: nerve impulse transmission

most membranes are multi-functional

~1/3 of all gene products in higher eukaryotes are membrane proteins

STRUCTURAL FEATURES OF MEMBRANES MULTIPLE COMPONENTS

lipids (phospholipids, glycolipids, cholesterol): bilayer structure forms main permeability barrier.

proteins (peripheral, integral): provide both structural and functional characteristics.

carbohydrate (covalently bound to lipid and protein): surface recognition.

BROAD COMPOSITIONAL VARIABILITYcorrelated with function

MOSTLY SELF ASSEMBLING

hydrophobic and electrostatic forces lead to bilayer formation and protein incorporation (carbohydrate added enzymically after assembly)

ASYMMETRIC

inside different from outside with respect to lipid and protein (carbohydrate only found on outer surface)

DYNAMIC STRUCTUREfluidity, flexibility, two-dimensional diffusion

STRUCTURES OF MEMBRANE LIPIDS

LIPID POLYMORPHISM

BIOLOGICAL SIGNIFICANCE OF LIPID POLYMORPHISM

potential to form nonbilayer structures may allow discontinuities in bilayer and thereby promote:

membrane fusion and vesicle formation during cell division.    vesicle-mediated protein trafficking.    integration of non-lipid components into membrane.    transport of macromolecules through membrane.    lateral movement of macromolecules.    stabilization of membrane protein complexes.    conformational interconversions associated with protein function.

MEMBRANE DYNAMICS

ELECTROSTATICS

TRANSLATIONAL (LATERAL) DIFFUSION IN MEMBRANES

usually measured by FRAP (fluorescence recovery after photobleaching) using fluorophore-labelled lipids.

Involves photobleaching a small region of membrane surface with laser and measuring time-dependence of molecular diffusion into bleached area.

Dtrans (translational diffusion coefficient) related to mean square displacement:

_r2 4 Dtrans t

for both lipids and proteins, Dtrans 10-8 cm2s-1 at 25 °C. Thus, in 1 second:

_ r2 = 4 x 10-8 cm2

_(r2)1/2 (mean displacement) = 2 x 10-4 cm = 2 microns (i.e. movement is rapid).

MEASUREMENT OF MEMBRANE FLUIDITY AND MOLECULAR ROTATION BY FLUORESCENCE DEPOLARIZATIONuse a covalently attached fluorophore, or a fluorescent probe which partitions into the bilayer (e.g. DPH; diphenylhexatriene). Excite with polarized light and measure polarization of fluorescence. If fluorophore rotates during excited state lifetime, fluorescence will become depolarized.

DEFINITIONS:P = polarization = (I - I) / (I + I)

r = anisotropy = (I - I) / (I + 2I)

PERRIN EQUATION:r0 / r = degree of depolarization = 1 + (F / C) 

WHERE:r0 = anisotropy in rigid matrix (i.e. no rotation)

r = anisotropy in membrane F = fluorescence lifetime

C = rotational correlation time = 1 / 6Drot

Drot = rotational diffusion coefficient

for a completely rigid system:r0 = 0.33 (when absorption and emission

dipoles are at 90°)r0 = 0.5 (when dipoles are parallel).

rotational diffusion coefficient is proportional to solvent viscosity:

Drot = kT/frot = kT/ 6 V (for spherical molecule)

where:frot = rotational frictional coefficient = viscosity V = volume of fluorophore

via Perrin equation, rotational correlation time can be related to solvent viscosity (for a spherical molecule) by: 

c = V / k T usually use a calibration curve to calculate microviscosity of medium. in general:  lipid bilayer 100 water

can also be applied to proteins in a membrane to obtain Drot ; for

two-dimensional rotational motion:Drot = k T / 4 a2 h (for a cylinder)

for a "typical" membrane protein: Drot = 105 s-1; c = 2 s

ha

c can be determined directly by measuring time dependence

of anisotropy.

ELECTROSTATIC EFFECTS AT MEMBRANE SURFACES membrane surface charge will influence local concentrations of charged species, including hydrogen ions, salt ions and proteins. the surface potential of a membrane can be calculated from electrostatic double layer theory (Goüy-Chapman theory; cf. Cevc & Marsh, “phospholipid bilayers”, Wiley-Interscience, 1987).

(in mV) = (2kT/Ze) ln (0.36 Ac C1/2) Z = charge valency of counterions Ac = surface charge density; area per charge at surface (in nm2) C = molar concentration of salt ions from this potential, one can calculate the local concentration of a charged protein, and the local pH:

[P]surface = [P]bulk exp(-Z / kT) where Z is the net protein charge.pHsurface = pHbulk + e / 2.3 kT

note that is always negative for biomembranes. Also, both of these quantities will be strongly affected by salt concentration.

HIGH-RESOLUTION MEMBRANE PROTEIN STRUCTURES

WEB SITE: blanco.biomol.uci.edu/Membrane_proteins_xtal.html

79 structures in pdb data base (as of March, 2004)

X-ray structures of membrane proteins (as of Jan. 2004)

Year

1986 1988 1990 1992 1994 1996 1998 2000 2002 2004

Num

ber

of s

truc

ture

s

0

2

4

6

8

10

12

14

16

18

20

22

24

CRYSTALLIZATION OF MEMBRANE PROTEINS

CURRENT OPINION IN STRUCTURAL BIOLOGY, 7, 697-701 (1997).Crystallization of membrane proteinsChristian Ostermeier and Hartmut Michel

“------ successes are partly based on advancesin the crystallization procedures for integral membraneproteins. Variation of the size of the detergent micelle and/orincreasing the size of the polar surface of the membraneprotein is the most important route to well-ordered membraneprotein crystals. The use of bicontinuous lipidic cubic phasesalso appears to be promising.-------”

CRYSTALLIZATION OF INTEGRAL MEMBRANE PROTEINS SOLUBILIZED IN DETERGENT MICELLES

crystals stabilized mainly by polar interactions between protein molecules and between detergent molecules.

detergent molecules must fit into crystal lattice; thus their size (smaller is better) and chemistry are important.

addition of small amphiphiles to crystallization medium often enhances crystal formation by replacing those detergent molecules that sterically interfere with lattice formation. Also, by making micelles smaller, they can allow better contact between polar surfaces of protein.

small amphiphiles also increase protein solubility.

SEE: NOLLER ET AL., FEBS LETT. 504, 179-186 (2001) FOR DISCUSSION OF MECHANISM OF CUBIC PHASE CRYSTALLIZATIONEHUD M. LANDAU AND JŰRG P. ROSENBUSCHProc. Natl. Acad. Sci. USAVol. 93, pp. 14532–14535, December 1996

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

“---- quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (‘‘seeding’’) and support growth by lateral diffusion of protein molecules in the membrane (‘‘feeding’’). ----- bacteriorhodopsin crystals diffracted to 3.7 Å resolution (NOW TO 1.6 )” (halorhodopsin and sensory rhodopsin II also crystallized in this way.)

MEMBRANE PROTEIN STRUCTURES

PNAS 99, 11055 (2002) Interactions between lipids and bacterial reaction centers determined by protein

crystallography Camara-Artigas, Brune, and Allen Department of Chemistry and Biochemistry and Center for the Study of Early Events in Photosynthesis, Arizona State University, “Three lipid molecules that lie on the surface of the protein are resolved in the electron density maps. ---cardiolipin ---phosphatidylcholine ---- glucosylgalactosyl diacylglycerol. ---- lipids are located in the hydrophobic region of the protein surface and interact predominately with hydrophobic amino acids, in particular aromatic residues. Although the cardiolipin is over 15 Å from the cofactors, the other two lipids are in close contact with the cofactors and may contribute to the difference in energetics for the two branches of cofactors that is primarily responsible for the asymmetry of electron transfer. The glycolipid is 3.5 Å from the active bacteriochlorophyll monomer and shields this cofactor from the solvent in contrast to a much greater exposed surface evident for the inactive bacteriochlorophyll monomer. The phosphate atom of phosphatidylcholine is 6.5 Å from the inactive bacteriopheophytin, and the associated electrostatic interactions may contribute to electron transfer rates involving this cofactor. Overall, the lipids span a distance of 30 Å, which is consistent with a bilayer-like arrangement suggesting the presence of an ‘‘inner shell’’ of lipids around membrane proteins that is critical for membrane function.”

Camara-Artigas et. al glycolipid

PC

cardiolipin

cardiolipin

glycolipid

PRINCIPLES OF MEMBRANE PROTEIN STRUCTURE

[Scarlata, "Membrane Protein Structure"; see also: White & Wimley, Ann. Rev. Biophys. Biomol. Struct. 28, 319 (1999); White, in “Membranes”, Biophysical Society on-line textbook].

   membrane protein environment is complex; it involves the aqueous region outside the membrane, electrical charges at the membrane surface, and the hydrophobic interior of the membrane. The steep dielectric gradient makes it highly unfavorable to bury a charge (20 kcal/mole) or have an unsatisfied H-bond (5 kcal/mole); this controls which residues incorporate within the membrane and which remain outside, as well as secondary and tertiary folding (-helices and -sheets favored; loops and random coils disfavored). lipid head groups can have strong electrostatic and H-bonding interactions with interfacial residues of a membrane protein.

   Hydrophobic thickness of the bilayer must match the hydrophobic length of the protein, e.g. transmembrane helix must be 18 residues long. Bilayer thickness may stabilize specific protein conformational states.

    Hydrocarbon chain packing may also stabilize specific protein structures; favors components which do not greatly disrupt their interactions; e.g., protein cylindrical shapes are preferred.

    Some generalizations: tertiary structures of membrane proteins have similar interior packing as soluble proteins; helices tilted 20 to facilitate packing between side chains; H-bonds between helices are rare and salt bridges rarely found. Because of helix dipoles, antiparallel arrangement of transmembrane helices preferred. Trp and tyr mainly present at membrane-water interfaces; act as "anchors".

SOME EXAMPLES OF MEMBRANE PROTEIN STRUCTURES

WILL FOCUS MAINLY ON STRUCTURES BUT WILLALSO BRIEFLY DESCRIBE FUNCTIONAL PROPERTIES

IN SOME CASES

Cu A

heme a

heme a3

Cu B

cyt c binding site

13 subunits

CCO COFACTORS

heme a

heme a3

Cu A

Cu B

Mg

distances:Cu A Fe a : 19Å

Cu A Fe a3 : 22Å

Fe a Fe a3 : 14Å

Mills & Ferguson-Miller, BBA 1555, 96-100 (2002)

PROTON TRANSPORT PATHWAYS IN CCO

TWO ROLES:1- GENERATION OF PMF2- REDUCTION OF OXYGEN TO WATER

                                                                           

Zhang et al., Nature 392, 677 (1998)

Cytochrome bc1

11 subunits dimer

Rieske protein2Fe-2S

Two conformationsobserved

FeS

                                                                           

Positions of redox centersin two conformationsof Rieske protein

Lange & HuntePNAS, 99, 2800(2002)

Cytochrome bc1-cytochrome c complex

KcsA Potassium Channel From Streptomyces lividans.  1BL8

                                                                                                                                                                         

tetramer ofidenticalsubunits

BACTERIORHODPSIN: a light-driven proton pump

R. viridisDeisenhofer, Michel and Huber

Light-harvesting pigmentfrom photosyntheticbacteria

McLuskey et al.Biochemistry 40,8783 (2001)

PROSTAGLANDIN H2 SYNTHASE-1

integral membrane protein, located primarily in the endoplasmic reticulum.

catalyzes the first committed step in prostaglandin biosynthesis (arachidonate to prostaglandin H2).

bifunctional: cyclooxygenase (target for NSAID’s: aspirin, ibuprofen, indomethacin); peroxidase

anchored to one leaflet of bilayer by amphipathic helices.

PROSTAGLANDIN H2 SYNTHASE

Prostaglandin H2 synthase.  1PRH

                                                                                                                                                                                               

PORINS

found in outer membranes of gram-negative bacteria.

form water-filled channels that allow the influx/outflux of small hydrophilic molecules.

have trimeric, beta-barrel structures; residues alternate between facing inward and outward. Thus, do not have long stretches of hydrophobic residues, as in transmembrane helices.

pores narrowed by inward folding of a loop into lumen of barrel. Have wide entrance and wide exit, and a short central constriction (about 10 deep and 10 wide). Minimizes frictional contact with walls, while still excluding large molecules.

Maltoporin Trimer From Salmonella typhimurium.  2MPR

                                                                                                                                                                                          

PORIN FROM RHODOBACTER CAPSULATUS

Weiss & Schulz,J. Mol. Biol. 227,493 (1992)

Alpha-hemolysin.  7AHL

                                                                                                                                                               

From Staph.aureus; heptameric pore-forming protein

Secreted as33 kD solubleprotein; aggregatesand insertsinto membrane.

Pore hashydrophobicexterior andhydrophilicinterior.

Song etal. Science,274, 1859(1996)

http://www.sciencemag.org/content/vol274/issue5294/images/large/se496452502b.jpeg

SEC Y – PROTEIN TRANSLOCATING COMPLEX

van denBerg et al.Nature 427,36 (2004)

heterotrimer

channel formsa passiveconduit forextendedpolypeptidechain

ELECTRON TRANSFER IN OXYGENICPHOTOSYNTHESIS

Khlbrandt,Nature 426,399 (2003).

x-ray structuresfor PSI and PSIIhave been determined

two light reactions bridge energy gap between water and NADPH

cyclic ET

complexes from algaeand cyanobacteria haveessentially the samestructures, despite largeevolutionary separation

cofactors (per monomer):4 hemes1 2Fe-2S cluster1 chlorophyll a1 β-carotene1 plastoquinone

no evidencefrom structuresfor movementof Rieske protein; however, presumed to occur because oflong distance between FeS and heme f (29 Å); hinge regioncontains many glycine residues

Kurisu et al. Science 302, 1009 (2003)

subunits:cyt b6

cyt fRieske ISPsubunit IVPetGPetLPetMPetN

Mol. wt. =217 kD

TDS: quinone analog inhibitor

central lipid-filled cavity

13 trans-membranehelices permonomer

space-filling view of cyt b6f

central cavity( 2 DOPC molecules bound)

note bound plastoquinone

TDS bound onp-side and PQon s-side;consistent withtransfer of PQ betweenmonomers

location of chlorophyll a (function unknown);no clear axial ligand can be seen (may be awater molecule bridging to a peptide carbonyl);edge exposed to lipid phase

location of β-carotene: near center oftransmembrane region; too far from chl ato quench triplet state (function unknown)

Environment of heme X (heme ci): covalently bound by one thioether linkage; has no axial amino acid ligand (1 water bound); located between heme bn and central cavity; note location of PQ

heme X mayfunction in cyclicelectron transfer(does not occurin cyt bc1); couldmediate flow ofelectrons betweenPQ and ferredoxin

ION TRANSPORT: POTASSIUM CHANNELS

KcsA from Streptomyces lividans: a diffusive K+ channel

Doyle et al. Science 280,69 (1998)

main questions:1- selectivity; K+ (radius1.33 Å) >104 Na+ (radius0.95 Å)2- high throughput;approaches diffusion limit( 108 per sec)

Streptococcuslividans KcsAchannel; tetramer of 4 identical subunits

3.2 Å resolution

each subunit has2 transmembranehelices; one faces the central pore and the other the lipid phase; sequence conservation among K+ channelsstrongest for poreregion and inner helix

pore helix(points towardcenter of cavity)

distribution of aromatic residues; form 2 layers nearmembrane-water interfaces; help to position channel in

membrane

cutaway view showing solvent-accessible surface and distribution of electrical charge

red: negative chargeblue: positive chargewhite: neutralyellow: hydrophobic

green spheres: K+ ions

note negativepotential nearentrance andexit and non-polarregion in between;this distribution ofcharge facilitateshigh throughput

view showing pore dimensions

total length 45 Å; selectivity filter is12 Å long (minimizesdistance over whichK+ interacts stronglywith channel)

diameter ofselectivity filtertoo narrow toaccommodate hydrated K+ ion;thus waters mustbe stripped off forion to enter

central cavityand followingregion largeenough to befilled with water

energy barrier to movement of ions through membrane is highest at the center; having water molecules in center and oriented helix dipoles compensates for this; hydrophobic lining of channel prevents ions from sticking to surface; having two ions in selectivity filter causes structure change that facilitates movement

conduction occurs when one ion enters from one side and a second exits from the other side; ions in selectivity filter repel each other and force one to move into central cavity; direction of flow determined by concentration gradient

K+ in central cavity is fullyhydrated [cf. Zhou et al. Nature 414, 43 (2001); also contains a discussion of the entry of K+ into the channel and of effect of K+ concentration on channel structure]

pore helices

side view of K+ ions in selectivity filter; note coordinationby carbonyl oxygens that closely mimic, and compensate for loss

of, waters of hydration

selectivity filter: composed of four evenly spaced layers of carbonyl oxygenatoms and one layer ofthreonine hydroxyl oxygens

Gly residuesallow all carbonyloxygens topoint in same direction

view from top through selectivity filter; note close fit and coordination of K+

by carbonyl oxygens

view down through selectivity filter showing networkof aromatic residues surrounding pore; acts to hold the

filter open to prevent accommodation of smaller Na+

KvAP from Aeropyrum pernix: a voltage-gated K+ channel

Jiang et al. Nature 423,33 (2003)

structure of KvaP – central pore isessentially identical to that of KscA;structures deviate beginning at theintracellular membrane leaflet

Fab fragments

KvaP – blueKscA - green

crystallized as antibodycomplex

gly gatinghinges

voltage sensor paddles (helices 3b and 4) – located on perimeter of pore and connected to main part of channel by 3a helix and sharp turn at helix 5; should allow free movement relative tochannel

close-up view of isolated voltagesensor paddle – 4 arg residues (117, 120, 123, 126) shown by mutation to be essential for gating (rest of sequence is hydrophobic); R133 forms salt bridge with D62, joining S4 and S2

comparison of structure ofisolated voltage paddle (b)with intact channel (a); provides evidence that paddleis highly flexible and is looselypacked against the pore;suggests that it acts as aseparate mobile domain(consistent with functionalvoltage-gated channel beingproduced by splicing a voltagepaddle with the KcsA channel)

voltage paddle

model for voltage-gating: + charges on paddle (arg residues) arecarried through the membrane by passage of action potential andact to open pore; consistent with experiments in which tetheredbiotin is accessible to avidin from the intracellular side when thechannel is closed and accessible from the extracellular side whenthe channel is open; mechanism implies that hydrophobic and electrostatic forces can balance each other to allow charges to bepulled through the membrane interior. Model still controversial.

MscL from Mycobacterium tuberculosis: a mechanicallygated K+ channel

structure determination: Chang et al. Science 282, 2220 (1998) – gated openby increase in lateral tension applied to the bilayer; non-selective; thought toprotect against osmotic stress; crystal structure corresponds to closed state

comparison of channel structures(note cytoplasmic domain of 5-helixbundle in MscL extending ~35 Å from membrane)

tetrameric

pentameric; threadedacross membrane inopposite manner toKcsA (N-terminal helixis inner); outer helicescontact inner helix ofadjacent subunit

gating mechanism: structure of open state still not known, buthigh non-selective conductance consistent with absence ofselectivity filter and suggests water-filled open pore diameter of ~ 40 Å.working model: lateral pressure in membrane clamps the channel in the closed state; rearrangements in the lipids packed around the channel in response to membrane stretching reduces the pressure and allows the channel to open via changes in helix tilt; role of extramembrane domain is uncertain (much of it can be removed without influencing function). (cf. Perozo and Rees, Curr. Opin. Struct. Biol. 13, 432 (2003) for additional discussion)

PROTON PUMPING BY BACTERIORHODOPSIN

PROTON TRANSPORT IN BACTERIORHODOPSIN

Simplest known example of a transmembrane ion pump; proton pumped from cytoplasm to outside; gradient utilized for ATP synthesis. Elucidation of mechanism of action resulted mainly from combination of crystallography and time-resolved spectroscopy (UV/vis; FTIR; resonance Raman; NMR).

structure: Luecke et al.J. Mol. Biol. 291, 899(1999).

MW ~ 24 kD7 transmembranehelices

N-terminus

C-terminus

retinal chromophoreattached to lys residuevia protonated Schiffbase

photoreaction causes isomerizationof 13,14 double bond from trans to cisconformation

H

H

BACTERIORHODOPSIN PHOTOCYCLE

proton release and proton uptake separated in time and space; protein relaxation much slower than photoisomerization

H+

H+

to extracellularmedium

fromcytoplasm

photoisomerization

From Neutze et al.BBA 1565, 144

(2002)

re-isomerization

internal proton transfer

from Mathies et al.Ann. Rev. Biophys.Biophys. Chem. 20,491 (1991).

Mechanism of photoisomerization

From Neutze et al.BBA 1565, 144 (2002)

SEQUENCE OF EVENTSIN PROTON TRANSPORTPATHWAY INBACTERIORHODOPSIN

transmembrane helices enclose a cavity that spans the membrane; retinal divides the cavity into two sections:1-extracellular (hydrophilic, wide); has H-bonded network of 4 residues (arg82, tyr57, glu194, glu204), and at least 6 bound water molecules. 2-cytoplasmic hydrophobic, narrow); contains only one residue involved in proton transport (asp96; has unusually high pK and is protonated in the dark), and fewer bound waters; has to undergo conformational changes during photocycle that allow water to enter.

DARK ADAPTED STATE

From Lanyi, on-linetextbook

active site can be thought of as consisting of a water molecule (W402) coordinated by the protonated retinal Schiff base, which is salt-linked to two anionic asp residues, asp85 and asp212. This charge pairing, plus additional H-bonds, stabilizes the buried charges. Schiff base is deprotonated during the photocycle.

buried chargesunusual for amembraneprotein

From Neutze et al.BBA 1565, 144 (2002)

view of cytoplasmic channel in dark-adaptedstate showing distorted α-helix G (so-called π-bulge)and single bound water

note H-bondbetween D96 and T46; raisespKa of D96,inhibiting protontransfer toSchiff base

EVENTS FOLLOWING LIGHT ABSORPTION

  Retinal photoisomerization is coupled to protein conformation changes. This is the result of a steric and electrostatic conflict of the chromophore with its binding site. Relaxation of this conflict drives the thermal (dark) reactions of the photocycle.

Proton is transferred from Schiff base to asp85 within about 50 s; helped by a small movement of helix C which brings them closer together. Proton may be derived directly from Schiff base (suggested that Schiff base pK decreases and asp pK increases due to changes in environment: Schiff base N moves to hydrophobic region and H-bonds form to carboxyl group), or indirectly from the bound water molecule, generating hydroxyl ion which removes proton from retinal.

Protonation states of asp85, glu204 and glu194 are linked. Transfer of proton to asp85 moves it away from retinal and allows movement of arg82 towards bottom of channel. This causes pK of glu204 to decrease; glu204 transfers proton to glu194, which causes proton release at the extracellular surface.

purple – darkyellow – M state

From Luecke et al.Science 286, 255 (1999)

note aromaticresiduesflanking retinal;these act toimmobilizepolyene chainand move inresponse tophotoisomerization;results in movementof helix F

isomerization ofretinal reversesdirection of Schiff base andcauses stericclash with W402

From Luecke et al.Science 286, 255 (1999)

STRUCTURAL CHANGESIN EXTRACELLULAR CHANNEL UPON FORMATION OF MSTATE

dark state

M state

loss of H-bondloss of W402

movement of R82, E194 and E204

Protein conformation change in M intermediate is caused by retinal isomerization; the 13-methyl group pushes on trp182, moving helix F. This opens cytoplasmic channel (aided by a local unwinding of helix G) and allows water to enter, which causes pK of asp96 to decrease; results in proton transfer to Schiff base.

DARK RE-ISOMERIZATION OF RETINAL Causes reversal of protein conformational change by removing steric clash with trp182. Restores the high pK of asp96, leading to reprotonation from cytoplasm.

Final proton transfer occurs from asp85 to glu204 (via arg82 and bound water molecule), thereby completing the photocycle.

SUMMARY OF OVERALL MECHANISM

Proton transport occurs via alternating access between Schiff base and the two membrane surfaces. Direction of transfer is controlled by pK changes caused by coupling between retinal photoisomerization and protein conformational changes.

Reprotonation of Schiff base from cytoplasm requires that pK of asp96 be lowered and proton pathway created (probably via bound water).

From Neutze et al.BBA 1565, 144 (2002)

structure of early M intermediate in mutant BR showing increased water content near asp96

and increased accessibility to Schiff base

protein conformation changes occurring during photocycle

local bend of helix C enables proton transfer to asp85

outward movementof helix F openscytoplasmicchannel, enablingwater to enter,proton transferto Schiff base, andreprotonation of asp96

structural relaxationrecovers originalconformation

From Lanyi and Schobert, Biochemistry 43, 3-8 (2004)

Retinal distorted due to counter-rotationsof C14-C15 and C5-N bonds; H-bond to water broken

Strained conformation in K storesfree energy; in subsequent steps retinal relaxes; coupled to proteinconformational changes.

Schiff base N-H realigns with water

Schiff base proton transferred to Asp-85

Schiff base N fully rotated to cytoplasmicside

Retinal fully bent

M1 to M2 is the reprotonation switch

RETINAL MOVEMENT DURINGFIRST HALF OF PHOTOCYCLE

MOVEMENT OF ARG-82 TOWARDS EXTRACELLULAR SURFACECAUSING PROTON RELEASE

FORMATION OF WATER CHAIN IN CYTOPLASMIC REGIONDURING SECOND HALF OF THE PHOTOCYCLE

13-methyl group pushes againstindole ring of Trp-182 in M2; Lys-216 side chain twists. Leads to repacking of side chains between helices F and G that results in outward tilt of helix F, inward tilt of helix G and formation of a cluster of water molecules between Asp-96 and Schiff base N.

pKa of Asp-96 is lowered causingreprotonation of Schiff base.