Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation.

ABSTRACT: Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including choice of site for ligation with retention of function, optimization of ligation yield, and purification from unreacted domains. Ligated sem

SUBMITTER: Clerico EM 

PROVIDER: S-EPMC2991546 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature