Structural Effects of Disease-Related Mutations in Actin-Binding Period 3 of Tropomyosin

Figure 1. Tpm1.1 and Tpm3.12 peptides design. (A) Schematic representation of the fragments with respect to the full-length model of Tpm (PDB ID: 1C1G). Sequence comparison of designed Tpm3.12 and Tpm1.1 fragments. The alignment was performed in UniProt, accession numbers P09493 for Tpm1.1 and P06753 for Tpm3.12. Stars between the sequences indicate identical amino acid residues, dots indicate sites where the sequences differ. Blue, orange, and magenta indicate positions of actin-binding periods 2, 3 and 4, respectively. Lower case italicized letters on top of the sequences show positions of the amino acid residues in heptad repeat and a and d positions are shown in cyan and red, respectively. Mutation sites related to hypo- and hypercontractile phenotypes are highlighted in yellow and red, respectively. The N-terminal fusion peptide (shaded with gray) comprises amino acid residues left after proteolysis of His-tag with thrombin, followed by Cys and elastic Gly-Gly linker introduced during peptide construction. (B) Illustration of the coiled-coil wheel where positions of the mutations are indicated by arrowheads.

Figure 2. SDS-polyacrylamide gel electrophoresis of the wild-type peptides under reducing and non-reducing conditions. Tpm1.164–154 (lanes 2–4) and Tpm3.1265–155 (lanes 5–7) separated on the SDS-gels after incubation for 3 min at 100 °C with Laemmli solution in the presence of 10% β-mercaptoethanol (lanes 2 and 5), in the absence of β-mercaptoethanol (lanes 3 and 6), or neither with boiling nor β-mercaptoethanol (lanes 4 and 7).

Figure 3. Effect of mutations on the thermal stability of Tpm1.164–154 and Tpm3.1265–155. Melting of Tpm1.164–154 (A), Tpm3.1265–155 (D), and their respective mutants (B,C,E,F) were measured at 222 nm in 100 mM NaCl, 10 mM Na-Phosphate, pH 7.0, with (○) and without (●) 0.5 mM DTT. Fraction unfolded is the ratio of unfolded to folded peptides (see Materials and Methods).

Figure 4. The R91P substitution in Tpm3.1265–155 results in decreased helicity in the presence of DTT. CD spectra were measured at 0 °C in 100 mM NaCl, 10 mM Na-Phosphate, pH 7.0, with or without 0.5 mM DTT.