Getting down and dirty with detergents: quantitation, screening, and synthesis

Philip D. Laible1, Samuel H. Gellman2, Deborah K. Hanson1, Christopher A. Kors1, Pil Seok Chae2, and Marc J. Wander1

1Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439

2Department of Chemistry, University of Wisconsin, Madison, WI 57306

Protein Structure Initiative “Bottlenecks” WorkshopNational Institutes of Health

Bethesda, MDApril 16, 2008

Membrane proteins: ultra important but difficult to study

Roughly 65:35 split between soluble and membrane-associated proteins in most genomes.

Cytoplasmic and periplasmic volume is 30 times greater than membrane volume inside a typical cell.

Membrane proteins are key to many processes and comprise the majority of drug targets.

Structural and functional studies are difficult as membrane proteins are hard to produce.

Relatively few structures.

NIH/DOE

Innermembrane

Cell wallPeriplasm

Outer membrane

Typical membrane protein production pipeline

Primary focus of Program Project

A strategy to produce membraneproteins for reagent and technology tests

Advantage of the Rhodobacter expression system: This organism can be engineered to provide coordinated synthesis of foreign membrane proteins with synthesis of new membrane into which they can be incorporated.

Invaginations of thecell membranefound in speciesof Rhodobacter

Model of Rhodobacter

cells underscoring key features

Electron micrographs of two Rhodobacter deletion strains

Laible et al., 2007

400 Rhodobacter expression constructs have been evaluated.

Overall Rhodobacter expression success is ~ 60%.

Genes representing entire membrane proteomes are being cloned into the Rhodobacter membrane protein expression system with 80% efficiency. Ligation-independent cloning enabled a significantly higher-throughput approach to the test for successful heterologous expression for this target set.

Efficiency of Cloning, Conjugation, and Expression

Current Statistics

Wes

tern

(ant

i-his)

Molecular Range of Expressed Membrane Proteins

100 75

50

30

15

Membrane Protein Production in Rhodobacter

Detergent Quantitation Detergent Screening Glycotripod amphiphiles

Enabling Technology/Reagent Short Story

Origin: Production core

Investigators:Chris KorsNick Impellitteri

Application:Production of well characterized/defined samples used throughout program.

We sought to develop a fast, inexpensive, and quantitative protocol to:

• create defined and reproducible membrane protein-detergent samples for input into structural and functional studies.

• facilitate replacement of detergents used for the solubilization and purification of a membrane protein with a diverse range of detergents that could potentially be more conducive to downstream characterization and crystallization attempts.

Measuring detergent levels inmembrane protein samples

Since determination of the detergent and lipid content of membrane protein samples can be:

• time consuming• expensive• cumbersome

Detergent Quantitation Detergent ExchangeTHIN LAYER CHROMATOGRAPHY

– Place chromatography paper and solvent in TLC tank.– Equilibrate for one hour. – Spot samples on TLC plate. – Place plate in sealed chamber, allow solvent migration.– Remove and thoroughly dry TLC plate.

IODINE VAPOR STAINING– Incubate desiccator in water bath (60C).– Add iodine crystals.– Seal and stain for no more than 15 minutes.

SCANNING AND QUANTIFICATION– Immediately scan plate.– Quantification of spot intensities.

CONCENTRATE

ON-COLUMN

Samples bound to column, washed with 1,

5, 10, or 20 column volumes (CV) of

replacement detergent buffer, and eluted.

DIALYSIS

Samples dialyzed for 1, 2, 5, or 7 days

Input: PURE PROTEIN1] Rhodobacter sphaeriodes Reaction Center (RC)2] Escherichia coli protein APC809 (thiol:disulfide interchange protein)

DETERGENTEXCHANGE

All detergents, except Triton X-100 and C8E4, displayed unique Rf values, which were not altered when the detergents were run as a mixture in the same lane.

A detergent “ladder” (L) was created to aid in the identification of detergent spots.

Visualizing Detergent ‘Spots’ on TLC Plates

Detergents and Detergent Ladder

PurificationDetergent

ReplacementDetergent

Example TLC Plate

Analysis of detergents as PDCs had no effect on expected Rf values as well (similar results obtained with other detergents).

Detergents and Detection Limits

For all detergents surveyed:

Linear standard curves were obtained.

Detection limit spanned well below both the CMC and the concentrations of the detergents in the buffers used in this study.

Samples of a wide range of concentrations were run on TLC plates and then quantified in order to determine the range of detection limits for each detergent.

Detergent Exchange by Dialysis is Incomplete

Dialysis NEVER allowed for complete detergent exchange; substantial residual amounts of purification detergent (LDAO) remained.

Amount of residual purification detergent scaled proportionally with CMC of replacement detergent.

– OG (highest CMC of all the exchanging detergent) yielded ONLY 50 % exchange after 7 days.

– Triton X-100 (lowest CMC of all the exchanging detergents) yielded 87% exchange after 7 days.

On-Column Detergent Exchange is Quantitative

On-column detergent exchange was faster, more definitive and reproducible compared to dialysis for ALL detergents tested.

All detergents but one were able to replace 100% of the purification detergent after washing with only 5 column volumes.

Experimental Details for Sample Analysis with MS

Don’t need state-of-the-art Mass Spec(although we used an LTQ-FT)

Poroshell 300SB-C3 column Water/Acetonitrile Gradient Injected 1 µl sample Each detergent had a unique retention time Generated standard curve using peak areas Limited range of concentrations where response is

linear

TLC results confirmed with Mass Spectrometry

Cost Comparison

Mass Spectometer:– Need efficient access; if not, acquisition costs astounding.– Method development alone can cost hundreds of dollars.– Individual sample runs are at least $50 (possibly > $100).

TLC with Iodine Vapor Staining:– Portable with minimal costs (once a desiccator, TLC tank, hot water bath, and a scanner were obtained). – The costs involved for chemicals, TLC plates, and chromatography paper were less than 50 cents per sample.

Average Exchange (%)TLC MS

Dialysis

On-column

69 18

98 2

49 15

97 0.3

Detergent Quantitation Detergent Screening Glycotripod amphiphiles

Enabling Technology/Reagent Short Story

Origin: Protein production core and detergent synthesis efforts

Investigators:Marc WanderAaron BowlingDeborah Hanson

Application:Discovery of new, generally useful, surfactants. Categorize known sets of detergents to make work with them less trial and error.

Detergent Selection

Detergent properties and micelle properties influence:

Yield of protein extracted from the lipid bilayer

Protein stability

Quantity and type of native lipids which are co-extracted with integral or membrane-associated proteins

Ultimately, functional properties and structural integrity

Crystallization propensity; thus, the solubilizing detergent may have to be exchanged before trials are initiated

a super-critical step in a purification scheme

Zhang et al, 2003

The ranking system focuses upon two important initial issuesin membrane protein purification:

The Detergent Screening Protocol

• Solubilization – tests ability of the detergent to disrupt the lipid bilayer and extract protein

• Stabilization – tests the ability of micelles of a detergent to stabilize the protein once removed from the membrane

• LHII is very stable, and therefore removed from our starting material

• LHI is very fragile and readily falls apart

• RCs are intermediate

The Screening Protocol: A Closer Look

Weak StrongWeak detergents extract complexes with LHI intact

Intermediate detergents break down LHI, RCs remain intact

Strong detergents break down LHI and RCs

Rhodobacter capsulatus strainutilized lacks LHII

LHI

RC

HT

Standardized protocol amenable to automation

Screening commences with homogenized Rhodobacter capsulatus membranes and proceeds on a relatively small scale in order to maximize the number of detergents that can be examined.

The Ranking System in Action

Strong surfactantLDAO

Intermediate surfactantTriton X-100, OG

Weak surfactantDDM, HEGA-11, CHAPS

Level 5Detergent

Level 3Detergent

Level 1Detergent

Weak Strong

+

Summary of Results

A total of 128 detergents have been investigated(e.g. Anatrace, Cognis, Sigma, Avanti Polar Lipids)

• Most detergents tested have a carbon chain length between 7 and 12 (broad range of extraction yield).

• Detergents with chain lengths <7 carbons have more consistent extraction success. Detergents with >12 carbons tend to have poor extraction.

• Maltosides tend to exhibit mild stabilizing characteristics (Levels 1 – 2).

• Glucosides are harsh, and dismantle the photosynthetic complex (Levels 3 – 5). • N-oxide groups display even harsher characteristics and are capable of dismantling the reaction center (Levels 3 – 6).

There are no correlations between:• Carbon chain length and protein stabilization.

• CMC and extraction/stabilization.

• Molecular weight and extraction/stabilization.

• Ionic nature and extraction/stabilization.

Some trends/patterns include:

How is this categorization planned to be used?

NEWProtein

Level 1Detergent

Level 2Detergent

Level 3Detergent

Level 4Detergent

Level 5Detergent

Level 6Detergent

Investigate a few detergent categories…

Examine 28 Level 1 Detergents

Examine 36 Level 2 Detergents

Examine 16Level 3 Detergents

Examine 9Level 4 Detergents

Examine 19Level 5 Detergents

Examine 9Level 6 Detergents

Determine which level detergent works best…

Explore only detergents in the desired class…

Examine 128 Detergents

Investigate ALL detergents…

NEWProtein

METHODMETHOD

1)

2)

…too time/material consuming!!

Detergent Quantitation Detergent Screening Glycotripod amphiphiles

Enabling Technology/Reagent Short Story

Origin: Detergent design and synthesis efforts

Investigators:Pil Seok ChaeSam Gellman

Application:Membrane protein solubilization, stabilization, and crystallization.

Design and Synthesis of Tripod Amphiphiles

a. Good solubility in aqueous media

b. Form micelles to extract the membrane proteins

Design of tripod amphiphiles

Synthesis of tripod amphiphiles

Exquisite balance between hydrophobicity and hydrophilicity

b. Scalable synthesis ( > 1.0 g): short steps and high yield

a. Facile structural modification by synthetic methods

c. High purity ( > 95 %) for reliable and definite results

c. Mild without denaturing of membrane protein complexes 8- Nonionic & zwitterionic amphiphiles vs. ionic amphiphiles

Quite efficient synthetic methods should be employed

Primary Detergents for Crystals of Membrane Proteins

Tripod amphiphile variants are being synthesized and evaluated

- +

HNO

NO

Hydrophobic moieties

Hydrophilic moieties

Nonionic ( glucose , maltose )Zwitterionic ( N- oxide )

O

O O

HOHO

OH

OH

OC12H25HOOH

HO

HO O

OH

OC8H17HOOH

Linear alkyl groups Nonlinear groups (aryl, cycloalkyl)

R

O

NH

O OHO

OH

HO OH

ONH

O OH

OHO

O

O

HO

HOOH

OH

OHOH

ONH

O

O

O

O OH

OOH

OOHOH

HO

OH

OHHO

HOOHOH

OH

ONH

OO O

OH OOH

HOOHOH

HOOH

ONH

OO OOH

HOO OHOH

HO

O O

OHO

OH O OH

OH

HO

OHHO OHOH

A five glycotripod amphiphile series

1

2

3

4

5

glucose

diglucose

triglucose

maltose

dimaltose

insoluble

high CMC

poor disruption

high CMC

poor disruption

TPA-2 rivals DDM for solubilization yieldmicelles are highly stabilizing (gentle)

Tripod versions of molecules 2 and 4 are superior to monopod version (tail replaced with C-12)

Saturated ring(TPA-2-S)

TPA-2-S is superior to DDM in this system.

Structural diversity among amphiphiles that efficiently extract and stabilize photosynthetic complexes from native membranes of R. capsulatus

O

NH

O

O

OH

OHO

OHHO

OHO

HO

OH

HO

I

O

NH

O

O

OH

OHO

OHHO

OHO

HO

OH

HO

O OO

HO

OHHO

OHO

HO

OHHO

NH

O

Note: Heavy Atom Incorporation

Note: Maltose Headgroup and Cyclization of Tripod Substituents

Note: Two Rings in Tripod

Tripod amphiphiles: selected recent developments

Acknowledgements

Program Project Members

Argonne National Lab– Deborah Hanson– Marc Wander– Aaron Bowling– Chris Kors– Nicholas Impelliterri

University of Wisconsin– Sam Gellman– Pil Seok Chae– Melissa Boersma

Outside Collaborations

University of Illinois – Chicago– Alex Schilling

Funding– NIH Roadmap Grant

• PO1 GM075913