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Remedial strategies in structural proteomics: Expression, purification, and crystallization of the vav1/rac1 complex

Academic Article
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Overview

authors

  • Brooun, A.
  • Foster, S. A.
  • Chrencik, J. E.
  • Chien, E. Y. T.
  • Kolatkar, A. R.
  • Streiff, M.
  • Ramage, P.
  • Widmer, H.
  • Weckbecker, G.
  • Kuhn, Peter

publication date

  • May 2007

journal

  • Protein Expression and Purification  Journal

abstract

  • The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich domain (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface compared to other GEF/GTPase pairs. Here, we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein interaction with potential to inhibit lymphocyte activation.

subject areas

  • Amino Acid Sequence
  • Cloning, Molecular
  • Crystallization
  • Cysteine
  • DNA, Complementary
  • Glutathione Transferase
  • Guanine Nucleotide Exchange Factors
  • Hydrolysis
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis
  • Nanotechnology
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Protein Structure, Tertiary
  • Proteins
  • Proteomics
  • Proto-Oncogene Proteins c-vav
  • Recombinant Proteins
  • Zinc
  • rac1 GTP-Binding Protein
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Research

keywords

  • cysteine-rich segment
  • guanine nucleotide exchange factor
  • protein-protein interaction
  • structural proteomics
  • surface mutagenesis
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Identity

PubMed Central ID

  • PMC1892187

International Standard Serial Number (ISSN)

  • 1046-5928

Digital Object Identifier (DOI)

  • 10.1016/j.pep.2006.10.027

PubMed ID

  • 17275330
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Additional Document Info

start page

  • 51

end page

  • 62

volume

  • 53

issue

  • 1

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