Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units

Coiled-coil domains mediate the oligomerization of many proteins. The assem bly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hyd rostatic pressure affects the equilibrium between denatured and native form s in the direction of the form that occupies a smaller volume. The hydropho bic core is the region more sensitive to pressure, which leads in most case s to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetam ide covalently bound to cysteine residues of tropomyosin (PIATm) and high h ydrostatic pressure to assess the chain interaction and the inherent instab ility of the coiled-coil molecule. The native and denatured states of tropo myosin were determined from the pyrene excimer fluorescence. The combinatio n of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-t emperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding . In contrast, under high pressure, unlabeled and labeled tropomyosin molec ules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the ide a that pressure produces subdomain denaturation and that the polypeptide ch ains do not separate. Substantial unfolding of tropomyosin was also verifie d by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different sus ceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.