title: Phosphorylation modulates the mechanical stability of the cardiac myosin-binding protein C motif.
abstract: Cardiac myosin-binding protein C (cMyBP-C) is a thick-filament-associated protein that modulates cardiac contractility through interactions of its N-terminal immunoglobulin (Ig)-like C0-C2 domains with actin and/or myosin. These interactions are modified by the phosphorylation of at least four serines located within the motif linker between domains C1 and C2. We investigated whether motif phosphorylation alters its mechanical properties by characterizing force-extension relations using atomic force spectroscopy of expressed mouse N-terminal cMyBP-C fragments (i.e., C0-C3). Protein kinase A phosphorylation or serine replacement with aspartic acids did not affect persistence length (0.43 ± 0.04 nm), individual Ig-like domain unfolding forces (118 ± 3 pN), or Ig extension due to unfolding (30 ± 0.38 nm). However, phosphorylation did significantly decrease the C0-C3 mean contour length by 24 ± 2 nm. These results suggest that upon phosphorylation, the motif, which is freely extensible in the nonphosphorylated state, adopts a more stable and/or different structure. Circular dichroism and dynamic light scattering data for shorter expressed C1-C2 fragments with all four serines replaced by aspartic acids confirmed that the motif did adopt a more stable structure that was not apparent in the nonphosphorylated motif. These biophysical data provide both a mechanical and structural basis for cMyBP-C regulation by motif phosphorylation.
authors: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont, USA.
authors: Arthur J
authors: AJ
authors: Michalek
authors: Jack W
authors: JW
authors: Howarth
authors: James
authors: J
authors: Gulick
authors: Michael J
authors: MJ
authors: Previs
authors: Jeffrey
authors: J
authors: Robbins
authors: Paul R
authors: PR
authors: Rosevear
authors: David M
authors: DM
authors: Warshaw
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001696:Biomechanical Phenomena; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D057446:Hydrodynamics; D008027:Light; D051379:Mice; D018625:Microscopy, Atomic Force; D008954:Models, Biological; D009994:Osmolar Concentration; D010766:Phosphorylation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship; D013696:Temperature; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D000099163:Myosin Binding Protein C