Screening and Identifying Membrane Proteins Favorable for Crystallization

Jared Kim1, Ho Leung Ng2

1 Department of Microbiology, University of Hawaii at Manoa, Honolulu, 2 Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.19
DOI:  10.1002/cpps.40
Online Posting Date:  November, 2017
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Abstract

This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n‐dodecyl‐β‐maltoside (DDM). Detergent extracts are then centrifuged, heat precipitated, and filtered to remove insoluble, thermally unstable, and/or aggregated proteins. Samples are then prepared for analysis by mass spectrometry: proteins are precipitated by methanol/chloroform extraction and subjected to reduction, alkylation, and protease digestion. The resulting peptides are passed through a detergent removal column, desalted, rehydrated in 0.1% formic acid (v/v), and identified by LC‐MS/MS. © 2017 by John Wiley & Sons, Inc.

Keywords: membrane proteins; protein crystallization; crystallography; mass spectrometry; proteomics

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells of interest (e.g., E. coli, Saccharomyces cerevisiae, etc.)
  • Terrific Broth (for E. coli; appendix 4A)
  • Yeast extract peptone dextrose medium (for S. cerevisiae; Difco)
  • Phosphate‐buffered saline (PBS), pH 7.5 (see recipe)
  • Lysis buffer with 1 M or 0.3 M NaCl (see recipe)
  • DNase I, dry powder
  • DNase I buffer, pH 7.5 (see recipe)
  • Detergent of interest [e.g. n‐dodecyl‐β‐maltoside (DDM); Anatrace, cat. no. D310]
  • Methanol, pre‐chilled on ice
  • Chloroform
  • 50 mM ammonium bicarbonate pH 7.5‐8.0
  • 6 M urea solution (see recipe)
  • 16 mM dithiothreitol (DTT)
  • 93.5 mM iodoacetamide (IAA)
  • 20 ng/µl trypsin, proteomics/mass spectrometry grade (e.g., AMRESCO or Promega)
  • StageTip Equilibration Buffer 1 (50% acetonitrile/50% water, v/v)
  • StageTip Equilibration Buffer 2 (0.1 % trifluoroacetic acid in water, v/v)
  • StageTip Wash Buffer (0.1% TFA/5% acetonitrile, in water, v/v)
  • StageTip Elution Buffer (0.1% formic acid in 95% acetonitrile, v/v)
  • 2.5‐liter shake flask
  • 50 ml conical tubes
  • Refrigerated centrifuge with both swinging‐bucket rotor and fixed‐angle rotor, capable of at least 50,000 × g
  • Probe sonicator
  • Ice and ice bucket
  • Teflon homogenizer
  • Micropipette tips
  • Biospec Bead‐Beater homogenizer
  • 0.4‐mm silica beads (for S. cerevisiae)
  • End‐over‐end rotator
  • 50° to 60°C water bath
  • Centrifugal concentrator 1‐MDa molecular weight cutoff (e.g., Vivaspin 500)
  • 1.5‐ml microcentrifuge tubes (1.5 ml)
  • Detergent removal spin column (e.g., Pierce, cat. no. 87776)
  • StageTips (Thermo Fisher Scientific)
  • Vacuum concentrator
  • Additional reagents and equipment for mass spectrometry (Chapter 16)
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Figures

Videos

Literature Cited

  Carpenter, E. P., Beis, K., Cameron, A. D., & Iwata, S. (2008). Overcoming the challenges of membrane protein crystallography. Current Opinion in Structural Biology, 18, 581–586. doi: 10.1016/j.sbi.2008.07.001.
  Galea, C. A., High, A., Obenauer, J. C., Mishra, A., Park, C. G., Punta, M., … Slaughter, C. A. (2009). Large‐scale analysis of thermo‐stable, mammalian proteins provides insights into the intrinsically disordered proteome. Journal of Proteome Research, 8, 211–226. doi: 10.1021/pr800308v.
  Hattori, M., Hibbs, R. E., & Gouaux, E. (2012). A fluorescence detection size‐exclusion chromatography‐based thermostability assay for membrane protein precrystallization screening. Structure, 20, 1293–1299. doi: 10.1016/j.str.2012.06.009.
  Kim, J., Kagawa, A., Kurasaki, K., Ataie, N., Cho, I. K., Li, Q. X., & Ng, H. L. (2015). Large‐scale identification of membrane proteins with properties favorable for crystallization. Protection Science, 24, 1756–1763. doi: 10.1002/pro.2766.
  Li, M., Hays, F. A., Roe‐Zurz, Z., Vuong, L., Kelly, L., Ho, C. M., … Stroud, R. M. (2009). Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening. Journal of Molecular Biology, 385, 820–830. doi: 10.1016/j.jmb.2008.11.021.
  Newby, Z. E. R., O'Connell, J. D., Gruswitz, F., Hays, F. A., Harries, W. E. C., Harwood, I. M., … Stroud, R. M. (2009). A general protocol for the crystallization of membrane proteins for X‐ray structural investigation. Nature Protocols, 4, 619–637. doi: 10.1038/nprot.2009.27.
  Prive, G. G. (2007). Detergents for the stabilization and crystallization of membrane proteins. Methods, 41, 388–397. doi: 10.1016/j.ymeth.2007.01.007.
  Prosinecki, V., Botelho, H. M., Francese, S., Mastrobuoni, G., Moneti, G., Urich, T., … Gomes, C. M. (2006). A proteomic approach toward the selection of proteins with enhanced intrinsic conformational stability. Journal of Proteome Research, 5, 2720–2726. doi: 10.1021/pr0602491.
  Russell, R. B., & Eggleston, D. S. (2000). New roles for structure in biology and drug discovery. Nature Structural Biology, 7(Suppl), 928–930. doi: 10.1038/80691.
  Slabinski, L., Jaroszewski, L., Rodrigues, A., Rychlewski, L., Wilson, I., Lesley, S., & Godzik, A. (2007). The challenge of protein structure determination—lessons from structural genomics. Protein Science, 16, 2472–2482. doi: 10.1110/ps.073037907.
  Tomasiak, T. M., Pedersen, B. P., Chaudhary, S., Rodriguez, A., Colmanares, Y. R., Roe‐Zurz, Z., … Stroud, R. M. (2001). General qPCR and plate reader methods for rapid optimization of membrane protein purification and crystallization using thermostability assays. Current Protocols in Protein Science, 77, 29.11:29.11.1–29.11.14.
  Wiener, M. C. (2004). A pedestrian guide to membrane protein crystallization. Methods, 34, 364–372. doi: 10.1016/j.ymeth.2004.03.025.
Internet Resources
  https://blanco.biomol.uci.edu/mpstruc
  White, S. Membrane proteins of known 3D structure.
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