Crystallization of Integral Membrane Proteins

Travis J. Barnard1, Jeremy L. Wally1, Susan K. Buchanan1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 17.9
DOI:  10.1002/0471140864.ps1709s47
Online Posting Date:  February, 2007
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Abstract

Over the last 20 years, the use of X‐ray crystallography has become a viable technique for the structure determination of integral membrane proteins. However, standard crystallizaton protocols must be modified to account for difficulties involved in handling membrane proteins, which arise primarily from having detergent present. This unit provides protocols that can be used to crystallize a purified membrane protein, including detergent exchange, sample concentration, initial screening using a crystallization robot, and finally, optimization of crystallization conditions to obtain diffraction‐quality crystals. These protocols were established for outer membrane proteins, but can be used for inner membrane proteins as well. Advice on alternative protocols, detergent selection, and optimization of crystallization conditions is provided.

Keywords: membrane protein; detergent; crystallization; robotics

     
 
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Table of Contents

  • Basic Protocol 1: Robotic Screening of Crystallization Conditions
  • Alternate Protocol 1: Manual Screening of Crystallization Conditions
  • Basic Protocol 2: Preparation of 24‐Well Plates for Optimization of Crystallization Conditions
  • Basic Protocol 3: Refinement of Initial Crystallization Conditions in 24‐Well Format
  • Support Protocol 1: Detergent Exchange by Size‐Exclusion Chromatography
  • Support Protocol 2: Preparation of Membrane Protein Sample for Crystallization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Robotic Screening of Crystallization Conditions

  Materials
  • 96‐condition screen (Table 17.9.1)
  • 96‐well U‐bottom microplates (Greiner Bio‐One)
  • 8‐ or 12‐channel multi‐channel pipet
  • Nanoliter pipetting robot (e.g., Mosquito, TTP LabTech)
  • 96‐well format crystallization sheets (e.g., CrystalClene Sheets HT, Molecular Dimensions)
  • Constant temperature incubators (e.g., Rumed, Molecular Dimensions)
    Table 7.9.1   Materials   Suggested 96‐Condition Screens for Initial Crystallization Trials of Membrane Proteins a   Suggested 96‐Condition Screens for Initial Crystallization Trials of Membrane Proteins

    Hampton ResearchCrystal Screen HTIndex HTSalt Rx HT
    Nextal BiotechnologiesThe Classics LiteThe PEGsThe AmSO4The AnionsThe CationsThe MPDsThe pHClearThe MbClass SuiteThe MbClass II Suite
    Molecular DimensionsMemstart/MemSys
    Emerald BiosystemsWizard Bloc (I&II)Cryo Bloc (I&II)OZMA 1‐4OZMA 8‐10

     aThese screens are listed by manufacturer in random order.

Alternate Protocol 1: Manual Screening of Crystallization Conditions

  Materials
  • 96‐condition commercial screens (Table 17.9.1)
  • 96‐well conical sitting‐drop microplates (Corning/Hampton Research)
  • 8‐ or 12‐channel multi‐channel pipet (50‐µl volume)
  • 8‐ or 12‐channel multi‐channel pipet (1‐µl volume)
  • 1‐µl repeater pipet
  • ClearSeal Film (Hampton Research)
  • Microplate centrifuge
  • Constant temperature incubators (e.g., Rumed, Molecular Dimensions)

Basic Protocol 2: Preparation of 24‐Well Plates for Optimization of Crystallization Conditions

  Materials
  • 10× salt stock solutions
  • 10× buffer stock solution
  • Precipitant stock solutions (concentrations will vary)
  • Cryoprotectants (e.g., glycerol), optional
  • 24‐well plates (e.g., VDX plates with sealant, Hampton Research)
  • Repeater pipet (optional)
  • Orbital platform shaker (optional)
  • 22‐mm siliconized square cover slides (Hampton Research)
  • Constant temperature incubators (e.g., Rumed, Molecular Dimensions)

Basic Protocol 3: Refinement of Initial Crystallization Conditions in 24‐Well Format

  Materials
  • Commercial screens with crystal formation (see protocol 1 or protocol 2)
  • 10× salt stock solutions
  • 10× buffer stock solution
  • Precipitant stock solutions (concentrations will vary)
  • 24‐well plates (e.g., VDX plates with sealant, Hampton Research)
  • Light microscope (4× to 20× magnification)
  • Hampton Additive Screen (optional)
  • 22‐mm siliconized square cover slides (Hampton Research)
  • Constant temperature incubators (e.g., Rumed, Molecular Dimensions)

Support Protocol 1: Detergent Exchange by Size‐Exclusion Chromatography

  Materials
  • Column buffer (see recipe)
  • Purified membrane protein sample (≥1 mg)
  • Sephacryl S300 HR 16/60 size exclusion column (GE Healthcare)
  • Äkta prime chromatography system (GE Healthcare) or equivalent
  • 15‐ml Centriprep concentrators (Millipore)
  • 0.22‐µm syringe filter

Support Protocol 2: Preparation of Membrane Protein Sample for Crystallization

  Materials
  • Detergent‐exchanged membrane protein sample (see protocol 5)
  • 15‐ml Centriprep concentrators (Millipore)
  • 500‐µl Microcon concentrators (Millipore)
  • 500‐µl Ultrafree 0.22‐µm filtration devices (Millipore)
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Figures

Videos

Literature Cited

   Chang, G., Spencer, R.H., Lee, A.T., Barclay, M.T., and Rees, D.C. 1998. Structure of the MscL homolog from Mycobacterium tuberculosis: A gated mechanosensitive ion channel. Science 282:2220‐2226.
   Dutzler, R., Campbell, E.B., and MacKinnon, R. 2003. Gating the selectivity filter in ClC chloride channels. Science 300:108‐112.
   Garavito, R.M., Picot, D., and Loll, P.J. 1996. Strategies for crystallizing membrane proteins. J. Bioenerg. Biomembr. 28:13‐27.
   Garman, E.F. and Doublie, S. 2003. Cryocooling of macromolecular crystals: Optimization methods. Methods Enzymol. 368:188‐216.
   Grisshammer, R. and Tate, C.G. 1995. Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28:315‐422.
   Iwata, S. ed. 2003. Methods and Results in Crystallization of Membrane Proteins. La Jolla, International University Line.
   le Maire, M., Champeil, P., and Møller, J.V. 2000. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim. Biophys. Acta 1508:86‐111.
   Lebowitz, J., Lewis, M.S., and Schuck, P. 2002. Modern analytical ultracentrifugation in protein science: A tutorial review. Protein Sci. 11:2067‐2079.
   Lemieux, M.J., Song, J., Kim, M.J., Huang, Y., Villa, A., Auer, M., Li, X.D., and Wang, D.N. 2003. Three‐dimensional crystallization of the Escherichia coli glycerol‐3‐phosphate transporter: A member of the major facilitator superfamily. Protein Sci. 12:2748‐2756.
   Locher, K.P., Lee, A.T., and Rees, D.C. 2002. The E. coli BtuCD structure: A framework for ABC transporter architecture and mechanism. Science 296:1091‐1098.
   Loll, P.J. 2003. Membrane protein structural biology: The high throughput challenge. J. Struct. Biol. 142:144‐153.
   McPherson, A. 1991. Useful principles for the crystallization of proteins. In Crystallization of Membrane Proteins. (H. Michel, ed.) pp. 1‐51. CRC Press, Boca Raton, Fla.
   McPherson, A. 1999. Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Michel, H. 1991. General and practical aspects of membrane protein crystallization. In Crystallization of Membrane Proteins. (H. Michel, ed.) pp. 73‐88. CRC Press, Boca Raton, Fla.
   White, S.H. 2004. The progress of membrane protein structure determination. Protein Sci. 13:1948‐1949.
   Wiener, M.C. 2004. A pedestrian guide to membrane protein crystallization. Methods 34:364‐372.
   Yue, W.W., Grizot, S., and Buchanan, S.K. 2003. Structural evidence for iron‐free citrate and ferric citrate binding to the TonB‐dependent outer membrane transporter FecA. J. Mol. Biol. 332:353‐368.
   Zulauf, M. 1991. Detergent phenomena in membrane protein crystallization. In Crystallization of Membrane Proteins. (H. Michel, ed.) pp. 53‐72. CRC Press, Boca Raton, Fla.
Internet Resources
   http://www.rcsb.org/pdb
  Protein data base—the site where all protein structures (determined by X‐ray crystallography, electron microscopy, and NMR) are deposited and made publicly available.
   http://www.mpibp‐frankfurt.mpg.de/michel/public/memprotstruct.html
  Hartmut Michel's listing of crystallized membrane proteins, including a statistical analysis of crystallization conditions and structure references.
   http://blanco.biomol.uci.edu/Membrane_Proteins_xtal.html
  Stephen White's listing of all solved membrane protein structures. This list is continuously updated and contains references to the individual structures.
   http://www.emeraldbiosystems.com/OnlineStore/home.php
  Web sites for crystallization instrumentation and reagents.
   http://www.hamptonresearch.com/
  The Anatrace Web site for the widest variety of high purity detergents for membrane protein purification and crystallization.
   http://www.moleculardimensions.com/us/index.ihtml
   http://www.nextalbiotech.com/
   http://www.anatrace.com/default.htm
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