Biochemistry Commons^™

Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch

Lila Gierasch

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 (in this case 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 …