Remedial strategies in structural proteomics: expression, purification, and crystallization of the Vav1/Rac1 complex
| Autor: | Gisbert Weckbecker, Ellen Y.T. Chien, Paul Ramage, Markus Streiff, Jill E. Chrencik, Alexei Brooun, Scott A. Foster, Peter Kuhn, Hans Widmer, Anand Kolatkar |
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| Rok vydání: | 2006 |
| Předmět: |
Proteomics
rac1 GTP-Binding Protein Protein Folding DNA Complementary Protein Conformation Molecular Sequence Data Plasma protein binding GTPase Biology Article Protein–protein interaction Protein structure Guanine Nucleotide Exchange Factors Nanotechnology Amino Acid Sequence Cysteine Cloning Molecular Proto-Oncogene Proteins c-vav Nuclear Magnetic Resonance Biomolecular Glutathione Transferase Zinc finger Hydrolysis Proteins Recombinant Proteins Protein Structure Tertiary Kinetics Zinc Biochemistry Mutagenesis Biophysics Protein folding Guanine nucleotide exchange factor Crystallization Biotechnology Protein Binding |
| Zdroj: | Protein expression and purification. 53(1) |
| ISSN: | 1046-5928 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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