Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Brett A. Colson, Andrew R. Thompson, L. Michel Espinoza-Fonseca, David D. Thomas

Research output: Contribution to journalArticlepeer-review

21 Scopus citations


We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electron- electron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiacmyosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility.

Original languageEnglish (US)
Pages (from-to)3233-3238
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number12
StatePublished - Mar 22 2016

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS: Experiments reported in this paper were performed at the Biophysical Technology Center, University of Minnesota Department of Biochemistry, Molecular Biology, and Biophysics. Norma Jimenez Ramirez and Benjamin Zeman provided technical support. Richard Moss (University of Wisconsin, Madison) provided mouse C0C2 cDNA. This study was supported by NIH Grant AR032961 (to D.D.T.), American Heart Association (AHA) Grant 12SDG12060656 (to L.M.E.-F.), and AHA Postdoctoral Fellowship 13POST17250009 and NIH Grant R00 HL122397 (to B.A.C.). This project made use of the outstanding high-performance computing facilities at the Minnesota Supercomputing Institute.


  • DEER
  • Fluorescence resonance energy transfer
  • Molecular dynamics simulation
  • Muscle
  • Protein kinase A


Dive into the research topics of 'Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics'. Together they form a unique fingerprint.

Cite this