Effects of the N-terminal Domains of Myosin Binding Protein-C in an in Vitro Motility Assay

Maria V Razumova,Justin F Shaffer,An-Yue Tu,Galina V Flint,Michael Regnier,Samantha P Harris

doi:10.1074/jbc.m606949200

Maria V Razumova, Justin F Shaffer + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m606949200

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Abstract

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Highlights

Myosin binding protein-C (MyBP-C)2 is a sarcomeric protein associated with the thick filaments of vertebrate striated muscle [1]
The idea that MyBP-C limits cross-bridge kinetics was initially proposed by Hofmann et al [6] who suggested that MyBP-C acts as an internal load within the sarcomere based on their observations that partial extraction of MyBP-C from skeletal fibers reversibly
The exact structural arrangement of MyBP-C within the sarcomere is not known, MyBP-C could contribute to an internal load by tethering myosin heads to the thick filament and thereby limiting the extension of attached myosin heads as shortening proceeds [6]

Summary

EXPERIMENTAL PROCEDURES

RhPh F-actin was added for 1 min followed by infusion of the motility buffer. RhPh actin filaments were followed with AB containing 100 nM each cTn and cTm. RhPh actin and regulatory proteins were reconstituted for 6 min before the motility buffer was added [16]. Three different flow cell loading sequences were used: 1) BSA (0.5 mg/ml, 3 min) 3 AB wash 3 motility buffer plus C1C2 and RhPh-actin. For experiments to assess ability of C1C2 to bind actin in the presence of regulatory proteins, flow cells were treated sequentially with C1C2 in AB (or AB buffer alone as control) followed by BSA. Cosedimentation Binding Assays—F-actin was prepared as described for in vitro motility experiments and dialyzed into AB buffer plus 1 mM ATP prior to use.

RESULTS

Fraction moving

DISCUSSION