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 Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta 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 delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), 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 PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), 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 Ca(2+), but stoichiometrically and at a different location in the complex than delta.
Priddy
Timothy S
TS
Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri 64110, USA.
Price
E Shane
ES
Johnson
Carey K
CK
Carlson
Gerald M
GM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011817:Rabbits
D002147:Calmodulin; D021122:Protein Subunits; D010764:Phosphorylase Kinase; D002118:Calcium