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Reference: Hochstrasser M and Funakoshi M (2012) Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes. Methods Mol Biol 832:349-62

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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 approximately 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.

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Journal Article
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Hochstrasser M, Funakoshi M
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