Combined pulsed electron double resonance EPR and molecular dynamics investigations of calmodulin suggest effects of crowding agents on protein structures

Andrew Stewart, Muralidharan Shanmugam, Roger Kutta, Nigel Scrutton, Janet Eleanor Lovett*, Sam Hay*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron–electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.
Original languageEnglish
Pages (from-to)1735–1742
Number of pages8
JournalBiochemistry
Volume61
Issue number17
Early online date18 Aug 2022
DOIs
Publication statusPublished - 6 Sept 2022

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