Abstract
Tropomyosin (Tpm) is an actin-binding coiled-coil protein. In muscle, it regulates contractions in a troponin/Ca2+-dependent manner and controls the thin filament lengths at the pointed end. Due to its size and periodic structure, it is difficult to observe small local structural changes in the coiled coil caused by disease-related mutations. In this study, we designed 97-residue peptides, Tpm1.164–154 and Tpm3.1265–155, focusing on the actin-binding period 3 of two muscle isoforms. Using these peptides, we evaluated the effects of cardiomyopathy mutations: I92T and V95A in Tpm1.1, and congenital myopathy mutations R91P and R91C in Tpm3.12. We introduced a cysteine at the N-terminus of each fragment to promote the formation of the coiled-coil structure by disulfide bonds. Dimerization of the designed peptides was confirmed by gel electrophoresis in the presence and absence of dithiothreitol. Using circular dichroism, we showed that all mutations decreased coiled coil stability, with Tpm3.1265–155R91P and Tpm1.164–154I92T having the most drastic effects. Our experiments also indicated that adding the N-terminal cysteine increased coiled coil stability demonstrating that our design can serve as an effective tool in studying the coiled-coil fragments of various proteins.
Highlights
Tropomyosin (Tpm) is a protein belonging to a family of actin regulatory proteins which bind to actin filaments to control interactions of actin with its binding partners
To limit the analyses to the conservative actin-binding period 3 and the sequences nearest to this period, we engineered 91-residue fragments of Tpm1.1 and Tpm3.12 comprising the entire 35-amino acid residue long actin-binding period 3 flanked by sequences of periods 2 and 4 at the N- and C-termini of period 3 (Figure 1)
The peptide carried an N-terminal fusion sequence with Cys residue separated from the Tpm sequence by a flexible Gly-Gly linker and GSHM tetrapeptide left after His-tag proteolysis
Summary
Tropomyosin (Tpm) is a protein belonging to a family of actin regulatory proteins which bind to actin filaments to control interactions of actin with its binding partners. Tpm1.1 and Tpm3.12 are highly homologous proteins showing 91% identity and ~96% sequence similarity [4], their regulatory functions are quantitatively differentiated [5,6]. It is not known, which sequence differences are responsible for the regulatory performance of the isoforms. Due to the α-helical fold of each Tpm chain, the hydrophobic residues are exposed on one face of the α-helix, which allows for locking them with the hydrophobic residues from the second chain in “knobs into holes” fashion This forms a hydrophobic core necessary for dimerization and stabilization of two chains in the coiled-coil structure. In addition to the stabilizing hydrophobic residues, the core of Tpm coiled coil harbors clusters of small hydrophobic or polar residues such as alanine and serine, which cause bending and local flexibility thought to be necessary for Tpm to wrap around actin filaments [7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
