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

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• High-resolution structural data is often unavailable for large protein complexes.
• Understanding subunit interactions and assembly is crucial for protein complex function.
• The eukaryotic 26S proteasome is a large, multi-subunit complex with incompletely understood organization.

Purpose of the Study:

• To provide detailed protocols for engineered cysteine cross-linking of yeast proteasome subunits.
• To enable determination of nearest-neighbor relationships and atomic-resolution interaction details.
• To investigate proteasome assembly intermediates and quaternary organization.

Main Methods:

• Engineered cysteine cross-linking for oxidative cross-linking between predicted polypeptide neighbors.
• Application of cross-linking in whole-cell yeast extracts.
• Cross-linking of purified active 26S proteasome complexes isolated by native polyacrylamide gel electrophoresis.
• Analysis of cross-linking in subcomplexes representing potential assembly intermediates.

Main Results:

• Demonstrated feasibility of engineered cysteine cross-linking for yeast proteasome subunits.
• Successfully mapped subunit proximities within the intact 26S proteasome.
• Provided insights into the composition of proteasome subcomplexes and assembly pathways.
• Generated atomic-resolution details of subunit interactions in the absence of high-resolution structural data.

Conclusions:

• Engineered cysteine cross-linking is a powerful method for studying large protein complexes like the 26S proteasome.
• The developed protocols facilitate the investigation of protein complex organization and assembly.
• This approach complements traditional structural biology methods when high-resolution data is limited.