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

The four integral δ 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 δ subunits transmit allosteric signals to PhK's remaining α, β, and γ subunits in activating the enzyme. Under mild conditions about 10% of the δ 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 δ to assess the conformational substates of PhKδ 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 PhKδ became less abundant in the presence of Ca2+, with the δ 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 PhKδ subunits were distinct from those of CaM-DA free in solution, ±Ca2+, as well as from exogenous CaM bound to the PhK complex as δ′. The distinction between δ and δ′ is that the latter binds only in the presence of Ca2+, but stoichiometrically and at a different location in the complex than δ.