Biomimetic reagents that promote membrane protein stability and crystallization

Transcription

1 Biomimetic reagents that promote membrane protein stability and crystallization Philip D. Laible 1, Samuel H. Gellman 2, Brian K. Kay 3, Deborah K. Hanson 1, and Peter K. Nollert 4 1 Biosciences Division, Argonne National Laboratory 2 Chemistry Department, University of Wisconsin Madison 3 Biological Sciences Department, University of Illinois Chicago 4 decde biostructures, Inc. NIH Roadmap Membrane Protein Production & Technologies Meeting La Jolla, CA November 1, 2007

2 Program Project rganization and Mission Three research projects enabling the generation of bio-inspired reagents for the stabilization of membrane proteins: Lipidic cubic phases that resemble a natural bilayer but allow for crystallization of membrane proteins (PI: Peter Nollert decde) Newly discovered or designed surfactant that replace lipid bilayers, mimicking their amphiphilic interactions with the membrane protein (PI: Samuel Gellman UW, Madison) Protein-based affinity reagents that interact with the target membrane protein, creating artificial protein-protein interactions that can enable structural and functional analyses (PI: Brian Kay UIC) Two core facilities provide materials and support for research projects: Protein Production (PI: Deborah Hanson ANL) Administration

3 Targets produced within the core facility Selection Criteria: An assay or means to assess structural and functional integrity exists Span three kingdoms, cover diverse structural classes Representative of large membrane protein sub-family High-resolution structure is unknown or was derived using protein purified from the native host Most are active as a monomer; some form homo-oligomeric or heterooligomeric complexes The function of the protein depends on the membrane-spanning portion. Divided into two classes: Benchmark proteins Protocols established Expression Purification Stabilization Reconstitution Characterization Quantities available immediately for protocol development More challenging targets produced heterologously End goal is not necessarily structure determination

4 A strategy to produce membrane proteins 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 Laible et al., 2007 Electron micrographs of two Rhodobacter deletion strains Model of Rhodobacter cells underscoring key features

5 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 Molecular Range of Expressed Membrane Proteins 15 Western (anti-his) Current Statistics 400 Rhodobacter expression constructs have been evaluated. verall Rhodobacter expression success is ~ 60%.

6 Enabling Technology/Reagent Short Story Detergent Exchange Impurity Tolerance Detergent Screening Glycotripod amphiphiles rigin: Production core Investigators: Chris Kors Nick Impelletteri Application: Production of well characterized/defined samples used throughout program.

7 Quantification of Detergent Exchange BACKGRUND: It has been perceived that detergent exchange could be accomplished by: Simple dialysis Extensively washing and eluting a column bound membrane protein sample with an alternate buffer containing a different, yet desired, detergent. The success of these approaches, however, has been poorly characterized. GALS: The goal of this study was to develop a fast, inexpensive, and quantitative protocol to: Replace 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. Create defined and reproducible membrane protein-detergent samples for input into structural and functional studies. Compare current detergent exchange techniques. Measure this inexpensive approach against more costly, but sensitive, mass spectrometry methods for detergent quantification.

8 Detergent Exchange Detergent Quantification Input: PURE PRTEIN 1] Rhodobacter sphaeriodes Reaction Center (RC) 2] Escherichia coli protein APC809 (thiol:disulfide interchange protein) THIN LAYER CHRMATGRAPHY 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. DIALYSIS N-CLUMN Samples dialyzed for 1, 2, 5, or 7 days DETERGENT EXCHANGE Samples bound to column, washed with 1, 5, 10, or 20 column volumes (CV) of replacement detergent buffer, and eluted. IDINE VAPR STAINING Incubate desiccator in water bath (60 C). Add iodine crystals. Seal and stain for no more than 15 minutes. SCANNING AND QUANTIFICATIN Immediately scan plate. Quantification of spot intensities. CNCENTRATE

9 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.

10 Visualizing Detergent Exchange on TLC Plates Detergents and Detergent Ladder Example TLC Plate Purification Detergent Replacement Detergent 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. Analysis of detergents as PDCs had no effect on expected R f values as well (similar results obtained with other detergents).

11 Detergent Exchange by Dialysis is Incomplete Dialysis NEVER allowed for complete detergent exchange; substantial residual amounts of purification detergent (LDA) remained. Amount of residual purification detergent scaled proportionally with CMC of replacement detergent. G (highest CMC of all the exchanging detergent) yielded NLY 50 % exchange after 7 days. Triton X-100 (lowest CMC of all the exchanging detergents) yielded 87% exchange after 7 days.

12 n-column Detergent Exchange is Quantitative n-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 Dialysis n-column Average Exchange (%) TLC MS 69 ± ± 2 49 ± ± 0.3 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 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 Is detergent analysis by TLC methods practical? Accuracy of the TLC plate method is comparable to mass spectrometry methods. Costs are minimal; methods are fast; sample requirements are small. Results confirm an overall trend of higher final concentrations of the original purification detergent in dialysis samples post-detergent exchange. Additional benefits of on-column exchange: It can be completed in a matter of minutes as opposed to dialysis which can take several days, a factor that can be very critical when studying proteins with a limited shelf life. It allows for the use of much smaller volumes of replacement detergent buffer than dialysis, reducing costs especially when employing more expensive detergents. ther benefits of TLC plate analysis: Technique takes only a few hours, allowing for the rapid assessment of membrane protein samples. Also allows for simultaneous visualization of bound and/or co-purifying lipids. Also readily illuminates how much detergents can be concentrated with protein during the final steps leading to the setup of crystallization experiments.

15 Enabling Technology/Reagent Short Story Detergent Exchange Impurity Tolerance Detergent Screening Glycotripod amphiphiles rigin: Work on LCP-based crystallization Investigators: Chris Kors Doug Davies Ellen Wallace Peter Nollert Liang Li Application: Rescue of samples that can never be purified to extent that is thought to be needed for crystallization trials.

16 Bacterial reaction centers readily crystallize using LCP-based approaches LCP formed as a 40:60 mixture of LDA-solubilized reaction centers and monoolein: including when LCP is contaminated with exogenous lipids 0% 0.1 % 0.5% 5 % 20 % Fraction of Polar Brain Lipids in Lipid Component

17 Head-to-Head Crystallization Trials Test three crystallization methods against a series of increasingly impure samples of bacterial reaction centers - Lipidic Cubic Phases (µl scale) - Sitting drop vapor diffusion (µl and nl scales) - Microcapillary crystallization (batch method on nl scale)

18 Reaction center samples of varied purity - Clean and dirty samples prepared, mixed to form intermediate samples - Impurity level monitored quantitatively by spectroscopy (A 280 /A 800 ratio)

19 Crystallization as a function of sample purity Numbers of crystals sharply declined for microcapillary and vapor diffusion Size of crystals was relatively unaffected by sample purity

20 Diffraction quality of LCP crystals independent of the purity of the RC sample A 280 /A A 280 /A A 280 /A LCP approaches may be the fastest way to obtain a crystal structure of a new target membrane protein as trials can begin before purification optimized LCP would be the only approach considered for samples which defy all polishing attempts.

21 Enabling Technology/Reagent Short Story Detergent Exchange Impurity Tolerance Detergent Screening Glycotripod amphiphiles rigin: 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.

22 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

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

24 The Screening Protocol: Solubilization 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.

25 The Ranking System in Action Weak surfactant DDM, HEGA-11, CHAPS Intermediate surfactant Triton X-100, G Strong surfactant LDA Level 1 Detergent Level 3 Detergent Level 5 Detergent + Weak Strong

26 Summary of Results A total of 128 detergents have been investigated (e.g. Anatrace, Cognis, Sigma, Avanti Polar Lipids) Some trends/patterns include: 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.

27 How is this categorization planned to be used? METHD 1) NEW Protein Investigate ALL detergents Examine 117 Detergents too time/material consuming!! Investigate a few detergent categories Level 1 Detergent Level 2 Detergent Determine which level detergent works best Explore only detergents in the desired class Examine 28 Level 1 Detergents Examine 36 Level 2 Detergents 2) NEW Protein Level 3 Detergent Level 4 Detergent Examine 16 Level 3 Detergents Examine 9 Level 4 Detergents Level 5 Detergent Examine 19 Level 5 Detergents Level 6 Detergent Examine 9 Level 6 Detergents

28 Enabling Technology/Reagent Short Story Detergent Exchange Impurity Tolerance Detergent Screening Glycotripod amphiphiles rigin: Detergent design and synthesis efforts Investigators: Pil Seok Chae Sam Gellman Application: Membrane protein solubilization, stabilization, and crystallization.

29 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

30 Primary Detergents for Crystals of Membrane Proteins

31 Tripod amphiphile variants are being synthesized and evaluated H H C 8 H 17 Nonionic ( glucose, maltose ) H H H H C 12 H 25 Zwitterionic ( N- oxide ) - N + Hydrophilic moieties HN Hydrophobic moieties R Linear alkyl groups Nonlinear groups (aryl, cycloalkyl) See Pil Seok Chae s poster (P10)

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

33 Acknowledgements Program Project Members Argonne National Lab Deborah Hanson Chris Kors Marc Wander Greg Tira Donna Mielke decde biostructures Peter Nollert Doug Davies Ellen Wallace Scott Lovell University of Wisconsin Sam Gellman Pil Seok Chae Melissa Boersma University of Illinois Chicago Brian Kay Zengping Hao Veronica Volgina utside Collaborations University of Chicago Rustem Ismagilov Liang Li University of Illinois Chicago Alex Schilling Synchrotron Facilities Advanced Photon Source Funding NIH Roadmap Grant P1 GM075913