Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca2+ sensitivity to the enzyme, but bind independently of Ca2+. In addition to binding Ca2+, an obligatory activator of PhK's phosphoryltransferase activity, the d subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the d subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor - acceptor pair (CaM-DA) was exchanged for d to assess the conformational substates of PhK delta by single molecule fluorescence resonance energy transfer (FRET) +/- Ca2+. The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca2+, as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKd became less abundant in the presence of Ca2+, with the d subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca2+-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKd subunits were distinct from those of CaM-DA free in solution, +/- Ca2+, as well as from exogenous CaM bound to the PhK complex as delta '. The distinction between delta and delta ' is that the latter binds only in the presence of Ca2+, but stoichiometrically and at a different location in the complex than delta.