Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 832)

Abstract

In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ∼2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative cross-linking between predicted polypeptide neighbors. Here, we provide detailed protocols for engineered cysteine cross-linking of yeast proteasome subunits in whole-cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates.

Key words

Disulfide engineering Proteasome Site-directed mutagenesis Nondenaturing polyacrylamide gel electrophoresis (native PAGE) Substrate overlay Saccharomyces cerevisiae Yeast Immunoblotting 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Molecular Biophysics and BiochemistryYale UniversityNew HavenUSA
  2. 2.Graduate School of Natural Science and TechnologyOkayama UniversityOkayamaJapan

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