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Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex
Author(s) -
Priddy Timothy S.,
Price E. Shane,
Johnson Carey K.,
Carlson Gerald M.
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.062747407
Subject(s) - chemistry , förster resonance energy transfer , allosteric regulation , phosphorylase kinase , calmodulin , protein subunit , stereochemistry , crystallography , fluorescence , molecule , conformational change , acceptor , enzyme , biophysics , glycogen phosphorylase , biochemistry , biology , physics , organic chemistry , quantum mechanics , gene , condensed matter physics
The four integral δ 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 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) ±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 PhKδ became less abundant in the presence of Ca 2+ , with the δ 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 PhKδ 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 δ′. The distinction between δ and δ′ is that the latter binds only in the presence of Ca 2+ , but stoichiometrically and at a different location in the complex than δ.