Site-directed oxidative protein crosslinking

Abstract

Many important biological processes are catalyzed by large multiprotein complexes. A central goal in the study of these “protein machines” is to characterize the organization of proteins and to determine if this organization changes during the catalytic cycle. For this purpose, methodology has been developed to deliver a latent crosslinking reagent to a particular protein in complex solutions. When activated, the crosslinking of the tagged protein to its nearest neighbors occurs without large-scale covalent modifications elsewhere in the complex. Recent technical advances in this methodology are described. The study of very large multiprotein complexes presents many technical challenges, including the elucidation of the organization of proteins and the overall architecture of the complex. Described in this report are improvements in a novel affinity crosslinking system developed recently, which allows the noncovalent delivery of an oxidative crosslinking reagent specifically to His 6-tagged proteins. This allows contacts made by the tagged protein to be studied in the context of large complexes and in complex mixtures such as crude extracts.