title: Disulfide engineering to map subunit interactions in the proteasome and other macromolecular complexes.
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.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. mark.hochstrasser@yale.edu
authors.forename: Mark
authors.initials: M
authors.lastname: Hochstrasser
authors.forename: Minoru
authors.initials: M
authors.lastname: Funakoshi
mesh_terms: D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015151:Immunoblotting / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000032:analysis* / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000737:chemistry; D016415:Sequence Alignment
chemical_list: D004220:Disulfides; D029701:Saccharomyces cerevisiae Proteins; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease