Structural and Functional Properties of Kappa Tropomyosin

Abstract

In the myocardium, the TPM1 gene expresses two isoforms of tropomyosin (Tpm), alpha (αTpm; Tpm 1.1) and kappa (κTpm; Tpm 1.2). κTpm is the result of alternative splicing of the TPM1 gene. We studied the structural features of κTpm and its regulatory function in the atrial and ventricular myocardium using an in vitro motility assay. We tested the possibility of Tpm heterodimer formation from α- and κ-chains. Our result shows that the formation of ακTpm heterodimer is thermodynamically favorable, and in the myocardium, κTpm most likely exists as ακTpm heterodimer. Using circular dichroism, we compared the thermal unfolding of ααTpm, ακTpm, and κκTpm. κκTpm had the lowest stability, while the ακTpm was more stable than ααTpm. The differential scanning calorimetry results indicated that the thermal stability of the N-terminal part of κκTpm is much lower than that of ααTpm. The affinity of ααTpm and κκTpm to F-actin did not differ, and ακTpm interacted with F-actin significantly worse. The troponin T1 fragment enhanced the κκTpm and ακTpm affinity to F-actin. κκTpm differently affected the calcium regulation of the interaction of pig and rat ventricular myosin with the thin filament. With rat myosin, calcium sensitivity of thin filaments containing κκTpm was significantly lower than that with ααTpm and with pig myosin, and the sensitivity did not differ. Thin filaments containing κκTpm and ακTpm were better activated by pig atrial myosin than those containing ααTpm.

Novel Mutation Glu98Lys in Cardiac Tropomyosin Alters Its Structure and Impairs Myocardial Relaxation

“…The cross-linking of E98K Tpm was achieved by several subsequent heatings of the sample directly in the measuring cell of the calorimeter in the absence of reducing agents, as described earlier [22]. The entirely cross-linked state of the E98K Tpm was achieved after the fifth heating of the sample (see Figure 3C).…”

“…After such a procedure, all Tpm samples were in an entirely reduced state [20]. The cross-linking of E98K Tpm was achieved by several subsequent heating-cooling procedures directly in the measuring cell of the calorimeter in the absence of reducing agents, as described earlier [22]. The thermal unfolding of Tpm species studied was fully reversible both in reduced and in cross-linked states, thus allowing deconvolution analysis of the heat sorption curves, i.e., their decomposition into separate thermal transitions (calorimetric domains).…”