Chemical Ligation and Isotope Labeling to Locate Dynamic Effects.

Abstract

Heavy isotope labeling of enzymes slows protein motions without disturbing the electrostatics and can therefore be used to probe the role of dynamics in enzyme catalysis. To identify the structural elements responsible for dynamic effects, individual segments of an enzyme can be labeled and the resulting effect on the kinetics of the reaction can be measured. Such hybrid isotopomers can be constructed by expressed protein ligation, in which complementary labeled and unlabeled peptide segments are prepared by recombinant gene expression and linked by means of chemical ligation. The construction of such hybrid isotopomers is exemplified here with the paradigmatic enzyme dihydrofolate reductase (DHFR) from Escherichia coli.