Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes.

Mark Hochstrasser, Minoru Funakoshi

Methods in molecular biology (Clifton, N.J.) 2012

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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.

Citation

Mark Hochstrasser, Minoru Funakoshi. Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes. Methods in molecular biology (Clifton, N.J.). 2012;832:349-62