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 assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature 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 molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.
Suarez
M C
MC
Programa de Biologia Estrutural, Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro, Brazil.
Lehrer
S S
SS
Silva
J L
JL
D000818:Animals; D003080:Cold Temperature; D005456:Fluorescent Dyes / Q000737:chemistry; D006358:Hot Temperature; D006874:Hydrostatic Pressure; D007460:Iodoacetamide / Q000031:analogs & derivatives* / Q000737:chemistry; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D014335:Tropomyosin / Q000737:chemistry*
D005456:Fluorescent Dyes; D014335:Tropomyosin; C029358:N-(1-pyrenyl)iodoacetamide; D007460:Iodoacetamide