Tropomyosin Exon 6b Is Troponin-specific and Required for Correct Acto-myosin Regulation

Abstract

The specificity of tropomyosin (Tm) exon 6b for interaction with and functioning of troponin (Tn) has been studied using recombinant fibroblast Tm isoforms 5a and 5b. These isoforms differ internally by exons 6a/6b and possess non-muscle exons 1b/9d at the termini, hence they lack the primary TnT 1 -tropomyosin interaction, allowing study of exon 6 exchange in isolation from this. Using kinetic techniques to measure regulation of myosin S1 binding to actin and fluorescently labeled Tm to directly measure Tn binding, we show that binding of Tn to both isoforms is similar (0.1-0.5 M) and both produce well regulated systems. Calcium has little effect on Tn binding to the actin⅐Tm complex and both exons produce a 3-fold reduction in the S1 binding rate to actin⅐Tm⅐Tn in its absence. This confirms previous results that show exon 6 has little influence on Tn affinity to actin⅐Tm or its ability to fully inhibit the acto-myosin interaction. Thin filaments reconstituted with Tn and Tm5a or skeletal Tm (containing exon 6b) show nearly identical calcium dependence of acto-myosin regulation. However, Tm5b produces a dramatic increase in calcium sensitivity, shifting the activation mid-point by almost an order of magnitude. This shows that exon 6 sequence and, hence, Tm structure in this region have a significant effect upon the calcium regulation of Tn. This finding supports evidence that familial hypertrophic cardiomyopathy mutations occurring adjacent to this region can effect calcium regulation.

Thermal unfolding of smooth muscle and nonmuscle tropomyosin α‐homodimers with alternatively spliced exons

“…The short 25-residue sequences encoded by exons 6b and 6a do not differ in stabilizing or destabilizing clusters defined by Hodges and coworkers in the hydrophobic core [35][36][37], both containing only one stabilizing cluster of five residues. However, sequence analysis of exon 6 using coiled-coil prediction software [38] suggests that the coiled-coil propensity of exon 6b (Tm5a) is lower than that of exon 6a (Tm5b) [7]. The DSC data presented are consistent with this prediction, showing that the replacement of exon 6b in Tm5a by exon 6a in Tm5b increases the thermal stability of the major thermal transition by 3.4°C.…”

“…The replacement of muscle‐specific exon 6b by nonmuscle exon 6a in recombinant rat smooth muscle α‐Tm was shown to increase the actin affinity of α‐Tm [5], and the same exon exchange has a similar effect between fibroblast Tm5a and 5b isoforms [6]. This replacement in rat fibroblast α‐Tm has been shown to increase the calcium sensitivity of the regulation of acto‐myosin interaction in the presence of troponin [7].…”

Structural Basis for Ca2+-regulated Muscle Relaxation at Interaction Sites of Troponin with Actin and Tropomyosin

“…This clash can be avoided by the induced conformational disruption of the N-terminal portion of the E helix of TnC and/or tropomyosin. 39,61 …”

Differential Actin-regulatory Activities of Tropomodulin1 and Tropomodulin3 with Diverse Tropomyosin and Actin Isoforms

“…Rabbit skeletal muscle ␣/␤TM was prepared as described previously (42). Recombinant rat TM5b (TM5b cDNA in the pJC20 vector), a gift from Michael A. Geeves (University of Kent, Canterbury, UK), was expressed in BL21 Escherichia coli and purified as described previously (43). A clone of human TM5NM1 was a gift from Andrea Bacconi (The Scripps Research Institute, La Jolla, CA) and amplified from a pEGFP-TM5NM1 expression vector using PCR primers designed to introduce NcoI and XhoI restriction sites for cloning into the bacterial pET-14b expression vector (Novagen).…”