Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy

Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with...

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Bibliographic Details
Published in:Biophysical journal 2011-10, Vol.101 (8), p.1968-1977
Main Authors: Karsai, Árpád, Kellermayer, Miklós S.Z., Harris, Samantha P.
Format: Article
Language:English
Subjects:
actin
Animals
atomic force microscopy
Binding sites
Biomechanical Phenomena
Carrier Proteins - chemistry
crosslinking
Disease
extensibility
fibronectins
Fibronectins - chemistry
immunoglobulins
Immunoglobulins - chemistry
Kinetics
Mechanical Phenomena
Mechanical properties
mechanical stress
Mice
Microscopy
Microscopy, Atomic Force
Muscle Contraction
Muscle, Motility, and Motor Proteins
myosin
Protein Stability
Protein Structure, Secondary
Protein Structure, Tertiary
Protein Unfolding
Proteins
recombinant proteins
sarcomeres
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Summary:Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2011.08.030