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

1. Getting down and dirty with detergents: quantitation, screening, and synthesis 1Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 2Department of Chemistry, University of Wisconsin, Madison, WI 57306 Philip D. Laible1, Samuel H. Gellman2, Deborah K. Hanson1, Christopher A. Kors1, Pil Seok Chae2, and Marc J. Wander1 Protein Structure Initiative “Bottlenecks” Workshop National Institutes of Health Bethesda, MD April 16, 2008

2. 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. Inner membrane Cell wall Periplasm NIH/DOE Outer membrane • 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.

3. Primary focus of Program Project Typical membrane protein production pipeline

4. Model of Rhodobacter cells underscoring key features Electron micrographs of two Rhodobacter deletion strains Laible et al., 2007 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 the cell membrane found in species of Rhodobacter

5. Molecular Range of Expressed Membrane Proteins 100 75 50 30 Western (anti-his) 15 Membrane Protein Production in Rhodobacter 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 • 400 Rhodobacter expression constructs have been evaluated. • Overall Rhodobacter expression success is ~ 60%.

6. Enabling Technology/Reagent Short Story • Detergent Quantitation • Detergent Screening • Glycotripod amphiphiles Origin: Production core Investigators: Chris Kors Nick Impellitteri Application: Production of well characterized/defined samples used throughout program.

7. 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

8. Detergent Quantitation Detergent Exchange THIN 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 (60C). • Add iodine crystals. • Seal and stain for no more than 15 minutes. SCANNING AND QUANTIFICATION • Immediately scan plate. • Quantification of spot intensities. Input: PURE PROTEIN 1] Rhodobacter sphaeriodes Reaction Center (RC) 2] Escherichia coli protein APC809 (thiol:disulfide interchange protein) DIALYSIS ON-COLUMN DETERGENT EXCHANGE Samples bound to column, washed with 1, 5, 10, or 20 column volumes (CV) of replacement detergent buffer, and eluted. Samples dialyzed for 1, 2, 5, or 7 days CONCENTRATE

9. Visualizing Detergent ‘Spots’ on TLC Plates Detergents and Detergent Ladder Example TLC Plate • 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. Purification Detergent Replacement Detergent • Analysis of detergents as PDCs had no effect on expected Rf values as well (similar results obtained with other detergents).

10. Detergents and Detection Limits 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. 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.

11. 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.

12. 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.

13. TLC results confirmed with Mass Spectrometry Average Exchange (%) TLC MS 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 49  15 Dialysis 69  18 On-column 98  2 97  0.3 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.

14. Enabling Technology/Reagent Short Story Origin: Protein production core and detergent synthesis efforts Investigators: Marc Wander Aaron Bowling Deborah Hanson Application: Discovery of new, generally useful, surfactants. Categorize known sets of detergents to make work with them less trial and error. • Detergent Quantitation • Detergent Screening • Glycotripod amphiphiles

15. Detergent Selection a super-critical step in a purification scheme 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 Zhang et al, 2003

16. The Detergent Screening Protocol The ranking system focuses upon two important initial issues in membrane protein purification: • 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

17. The Screening Protocol: A Closer Look HT Rhodobacter capsulatus strain utilized lacks LHII • LHII is very stable, and therefore removed from our starting material • LHI is very fragile and readily falls apart • RCs are intermediate Strong Weak Weak detergents extract complexes with LHI intact Intermediate detergents break down LHI, RCs remain intact Strong detergents break down LHI and RCs

18. 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.

19. The Ranking System in Action Strong surfactant LDAO Weak surfactant DDM, HEGA-11, CHAPS Intermediate surfactant Triton X-100, OG Level 1 Detergent Level 3 Detergent Level 5 Detergent + Strong Weak

20. Summary of Results A total of 128 detergents have been investigated (e.g. Anatrace, Cognis, Sigma, Avanti Polar Lipids) • Carbon chain length and protein stabilization. • CMC and extraction/stabilization. • Molecular weight and extraction/stabilization. • Ionic nature and extraction/stabilization. Some trends/patterns include: There are no correlations between: • 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).

21. How is this categorization planned to be used? METHOD Investigate ALL detergents… NEW Protein 1) Examine 128 Detergents …too time/material consuming!! Investigate a few detergent categories… Explore only detergents in the desired class… Determine which level detergent works best… Level 1 Detergent Examine 28 Level 1 Detergents Level 2 Detergent Examine 36 Level 2 Detergents NEW Protein Level 3 Detergent Examine 16 Level 3 Detergents 2) Level 4 Detergent Examine 9 Level 4 Detergents Level 5 Detergent Examine 19 Level 5 Detergents Examine 9 Level 6 Detergents Level 6 Detergent

22. Enabling Technology/Reagent Short Story Origin: Detergent design and synthesis efforts Investigators: Pil Seok Chae Sam Gellman Application: Membrane protein solubilization, stabilization, and crystallization. • Detergent Quantitation • Detergent Screening • Glycotripod amphiphiles

23. Design and Synthesis of Tripod Amphiphiles Design of tripod amphiphiles a. Good solubility in aqueous media b. Form micelles to extract the membrane proteins c. Mild without denaturing of membrane protein complexes 8- Nonionic & zwitterionic amphiphiles vs. ionic amphiphiles Exquisite balance between hydrophobicity and hydrophilicity Synthesis of tripod amphiphiles a. Facile structural modification by synthetic methods b. Scalable synthesis ( > 1.0 g): short steps and high yield c. High purity ( > 95 %) for reliable and definite results Quite efficient synthetic methods should be employed

24. Primary Detergents for Crystals of Membrane Proteins

25. O H O H O O H H O H O O O O C H H O 1 2 2 5 O H H N Linear alkyl groups Nonlinear groups (aryl, cycloalkyl) Tripod amphiphile variants are being synthesized and evaluated Nonionic ( glucose , maltose ) Zwitterionic ( N- oxide ) + - O N Hydrophilic moieties O Hydrophobic moieties

26. Saturated ring (TPA-2-S) high CMC poor disruption high CMC poor disruption A five glycotripod amphiphile series Tripod versions of molecules 2 and 4 are superior to monopod version (tail replaced with C-12) glucose insoluble TPA-2 rivals DDM for solubilization yield micelles are highly stabilizing (gentle) 1 diglucose 2 triglucose 3 4 maltose TPA-2-S is superior to DDM in this system. dimaltose 5

27. Tripod amphiphiles: selected recent developments Structural diversity among amphiphiles that efficiently extract and stabilize photosynthetic complexes from native membranes of R. capsulatus Note: Two Rings in Tripod Note: Heavy Atom Incorporation Note: Maltose Headgroup and Cyclization of Tripod Substituents

28. 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