Eleven nearly invariant residues cluster around the active-site cleft, mostly interacting with the phosphates of ATP or facilitating phosphoryl transfer. Opening and closing of the active-site cleft is thus an essential feature of catalysis. Tethering it in any way will render the enzyme inactive. The crystal structures provide a static view of various conformational states, but understanding how the domains move in solution and how those motions correlate with functional steps of catalysis requires solution methods. We engineered a series of mutant proteins in which cysteines are selectively introduced and then labeled with a fluorescent probe.
Fluorescence anisotropy provides a tool for measuring local motion.
These mutant enzymes not only allow us to map different surfaces but also help us to understand the intricate network of interacting residues that link the active site to other regions of the protein. We recently showed that PKA, like AKT, can be phosphorylated on its activation loop Thr by the phospholipid-dependent protein kinase, PDK1. The structural changes that occur as a consequence of this phosphorylation are being explored. RI a , provided a first glimpse of the tandem cAMP-binding domains. This is an ancient signaling motif conserved from bacteria to humans.