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Probability analysis of variational crystallization and its application to gp120, the exterior envelope glycoprotein of type 1 human immunodeficiency virus (hiv-1)

Academic Article
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Overview

authors

  • Kwong, P. D.
  • Wyatt, Richard
  • Desjardins, E.
  • Robinson, J.
  • Culp, J. S.
  • Hellmig, B. D.
  • Sweet, R. W.
  • Sodroski, J.
  • Hendrickson, W. A.

publication date

  • 1999

journal

  • Journal of Biological Chemistry  Journal

abstract

  • The extensive glycosylation and conformational mobility of gp120, the envelope glycoprotein of type 1 human immunodeficiency virus (HIV-1), pose formidable barriers for crystallization. To surmount these difficulties, we used probability analysis to determine the most effective crystallization approach and derive equations which show that a strategy, which we term variational crystallization, substantially enhances the overall probability of crystallization for gp120. Variational crystallization focuses on protein modification as opposed to crystallization screening. Multiple variants of gp120 were analyzed with an iterative cycle involving a limited set of crystallization conditions and biochemical feedback on protease sensitivity, glycosylation status, and monoclonal antibody binding. Sources of likely conformational heterogeneity such as N-linked carbohydrates, flexible or mobile N and C termini, and variable internal loops were reduced or eliminated, and ligands such as CD4 and antigen-binding fragments (Fabs) of monoclonal antibodies were used to restrict conformational mobility as well as to alter the crystallization surface. Through successive cycles of manipulation involving 18 different variants, we succeeded in growing six different types of gp120 crystals. One of these, a ternary complex composed of gp120, its receptor CD4, and the Fab of the human neutralizing monoclonal antibody 17b, diffracts to a minimum Bragg spacing of at least 2.2 A and is suitable for structural analysis.

subject areas

  • Antibodies, Monoclonal
  • Antigens, CD4
  • Crystallization
  • Glycosylation
  • HIV Envelope Protein gp120
  • HIV-1
  • Humans
  • Protein Conformation
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.274.7.4115

PubMed ID

  • 9933605
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Additional Document Info

start page

  • 4115

end page

  • 4123

volume

  • 274

issue

  • 7

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