The local and global unfolding of coiled-coil tropomyosin.
The thermal unfolding of a two-stranded alpha-helical coiled coil of tropomyosin was studied using circular dichroism and excimer fluorescence of N-(1-pyrenyl)iodoacetamide-labeled tropomyosin. Tropomyosin unfolds with two transitions, namely local and global unfolding at high salt (greater than 0.1 M NaCl) and pH 7.5. The local unfolding was masked by the global unfolding at low salt (less than 0.1 M NaCl), at high pH (greater than pH 9.0), and in the presence of methanol, where the global unfolding temperature was similar to or lower than the local unfolding temperature. The local and global unfolding are different in nature. A comparison of the helix thermal unfolding of N-(1-pyrenyl)iodoacetamide-tropomyosin with unlabeled tropomyosin showed that tropomyosin had an inherent less-stable region, when Cys190 was N-(1-pyrenyl)iodoacetamide-labeled, disulfide cross-linked, or reduced. Instead, the chemical state of Cys190, determined the stability of the local unfolding region, because a strain created by the disulfide cross-link or the pyrene/pyrene interaction decreases the stability of the local unfolding region. Thus, these data show that the excimer fluorescence of N-(1-pyrenyl)iodoacetamide-labeled tropomyosin is useful for studying the local and global unfolding of tropomyosin.
Ishii
Y
Y
Department of Muscle Research, Boston Biomedical Research Institute.
D000818:Animals; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000031:analogs & derivatives; D000432:Methanol / Q000494:pharmacology; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014335:Tropomyosin / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology
D005456:Fluorescent Dyes; D014335:Tropomyosin; D012965:Sodium Chloride; C029358:N-(1-pyrenyl)iodoacetamide; D014508:Urea; D000432:Methanol; D007460:Iodoacetamide