Modeling Membrane Proteins Utilizing Information from Silent Amino Acid Substitutions

Uzi Kochva1, Hadas Leonov1, Isaiah T. Arkin1, Paul D. Adams2

1 The Hebrew University, Jerusalem, 2 Lawrence Berkeley Laboratory, Berkeley, California
Publication Name:  Current Protocols in Bioinformatics
Unit Number:  Unit 5.3
DOI:  10.1002/0471250953.bi0503s04
Online Posting Date:  February, 2004
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This unit describes predicting the structure of simple transmembrane α‐helical bundles. The protocol is based on a global molecular dynamics search (GMDS) of the configuration space of the helical bundle, yielding several candidates structures. The correct structure amongst these candidates is selected using information from silent amino acid substitutions, employing the following premise: Only the correct structure must (by definition) accept all of the silent amino acid substitutions. Thus, the correct structure is found by repeating the GMDS for several close homologues and selecting the structure that persists in all of the trials.

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Table of Contents

  • Basic Protocol 1: Selecting a Correct Protein Structure Using CHI
  • Guidelines for Understanding Results
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Selecting a Correct Protein Structure Using CHI

  Necessary Resources
  • Hardware
    • Hardware requirements are defined by those that are officially supported by CNSsolve, i.e., one of the following computers:
      • SGI (R4000 and later) running IRIX 4.0.5 or later
      • HP (PA Risc) running HP‐UX 9.05 or later
      • DEC Alpha running OSF1/Digital Unix/Tru64 Unix
      • PC (i386, i486, i586, or i686) running Linux or Windows 98 or NT or higher
    • Additionally, CNSsolve also provides unsupported installations for other systems:
      • Convex running ConvexOS:
      • Cray (J90, YMP, C90, T90) running Unicos
      • Cray T3E (single CPU) running Unicosmk
      • IBM RS/6000 running AIX
      • Sun running SunOS
      • Unix systems with g77/gcc (EGCS‐1.1)
      • Windows 98 or NT (or higher) systems with g77/gcc (EGCS‐1.1)
    • A Macintosh OS X port is also available (contact the authors for details; )
  • Software
    • CNSsolve: available free of charge for academic users at
    • CHI: available from Paul D. Adams ( )
    • Perl: Perl is a component of nearly all standard Unix distributions. It is available free of charge at Install according to the instructions on the Web page.
    • Three Perl scripts: (1) ak, (2), and (3) to from the authors; )
    • A CNSsolve input script, cns.inp (available from the authors; )
    • A standard text editor, e.g., jot, notepad, or nedit)
    • A Web browser
    • Software to perform multiple sequence alignment (e.g., ClustalX, ClustalW, or Pileup from the GCG Wisconsin package)
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Literature Cited

   Adams, P.D., Arkin, I.T., Engelman, D.M., and Brünger, A.T. 1995. Computational searching and mutagenesis suggest a structure for the pentameric transmembrane domain of phospholamban. Nat. Struct. Biol. 2:154‐162.
   Arkin, I.T. 2002. Structural aspects of oligomerization taking place between the transmembrane alpha‐helices of bitopic membrane proteins. Biochim. Biophys. Acta 1565:347‐363.
   Arkin, I.T., Adams, P.D., MacKenzie, K.R., Lemmon, M.A., Brünger, A.T., and Engelman, D.M. 1994. Structural organization of the pentameric transmembrane alpha‐helices of phospholamban, a cardiac ion channel. EMBO J. 13:4757‐4764.
   Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse‐Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T., and Warren, G.L. 1998. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54:905‐921.
   Kukol, A., Adams, P.D., Rice, L.M., Brünger, A.T., and Arkin, T.I. 1999. Experimentally based orientational refinement of membrane protein models: A structure for the Influenza A M2 H+ channel. J. Mol. Biol. 286:951‐962.
   Kukol, A., Torres, J., and Arkin, I.T. 2002. A structure for the trimeric MHC class II‐associated invariant chain transmembrane domain. J. Mol. Biol. 320:1109‐1117.
   Lemmon, M.A., Flanagan, J.M., Hunt, J.F., Adair, B.D., Bormann, B.J., Dempsey, C.E., and Engelman, D.M. 1992a. Glycophorin A dimerization is driven by specific interactions between transmembrane alpha‐helices. J. Biol. Chem. 267:7683‐7689.
   Lemmon, M.A., Flanagan, J.M., Treutlein, H.R., Zhang, J., and Engelman, D.M. 1992b. Sequence specificity in the dimerization of transmembrane alpha‐helices. Biochemistry 31:12719‐12725.
   Lemmon, M.A., Treutlein, H.R., Adams, P.D., Brünger, A.T., and Engelman, D.M. 1994. A dimerization motif for transmembrane alpha‐helices. Nat. Struct. Biol. 1:157‐163.
   MacKenzie, K.R., Prestegard, J.H., and Engelman, D.M. 1997. A transmembrane helix dimer: Structure and implications. Science 276:131‐133.
   Rice, L.M. and Brünger, A.T. 1994. Torsion angle dynamics: Reduced variable conformational sampling enhances crystallographic structure refinement. Proteins 19:277‐290.
   Stevens, T.J. and Arkin, I.T. 2000. Do more complex organisms have a greater proportion of membrane proteins in their genomes? Proteins 39:417‐420.
   Torres, J., Adams, P.D., and Arkin, I.T. 2000. Use of a new label, 13C=18O, in the determination of a structural model of phospholamban in a lipid bilayer: Spatial restraints resolve the ambiguity arising from interpretations of mutagenesis data. J. Mol. Biol. 300:677‐685.
   Torres, J., Kukol, A., and Arkin, I.T. 2001. Mapping the energy surface of transmembrane helix‐helix interactions. Biophys. J. 81:2681‐2692.
   Torres, J., Briggs, J.A., and Arkin, I.T. 2002a. Contribution of energy values to the analysis of global searching molecular dynamics simulations of transmembrane helical bundles. Biophys. J. 82:3063‐3071.
   Torres, J., Briggs, J.A., and Arkin, I.T. 2002b. Convergence of experimental, computational and evolutionary approaches predicts the presence of a tetrameric form for CD3‐zeta. J. Mol. Biol. 316:375‐384.
   Torres, J., Briggs, J.A., and Arkin, I.T. 2002c. Multiple site‐specific infrared dichroism of CD3‐zeta, a transmembrane helix bundle. J. Mol. Biol. 316:365‐374.
   Treutlein, H.R., Lemmon, M.A., Engelman, D.M., and Brü Nger, A.T. 1992. The glycophorin A transmembrane domain dimer: Sequence‐specific propensity for a right‐handed supercoil of helices. Biochemistry 31:12726‐12732.
Key References
   Arkin et al., 1994. See above.
  In this article, global searching molecular dynamics simulation is used to find a model for phospholamban.
   Adams et al., 1995. See above.
  Here, the theory of global searching molecular dynamics simulation is presented in detail.
   Briggs, J.A.G., Torres, J., Kukol, A., and Arkin, I.T. 2001. A new method to model membrane protein structure based on silent amino‐acid substitutions. Proteins Struct. Funct. Genet. 44:370‐375.
  In this article, silent substitution modeling is introduced for the first time.
   Torres et al., 2002a. See above.
  In this paper, results of global searching molecular dynamics simulations are analyzed in terms of energy, thereby enabling the user to further select among candidate models.
   Torres et al., 2002b. See above.
  In this work, silent substitution modeling is employed to derive a structure of the TCR CD3ζ transmembrane helical bundle, shown to coincide with that obtained experimentally.
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