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CD and 13 C α ‐NMR studies of folding equilibria in a two‐stranded coiled coil formed by residues 190–254 of α‐tropomyosin
Author(s) -
Holtzer Marilyn Emerson,
Mints Lisa,
Angeletti Ruth Hogue,
d'Avig D. André,
Holtzer Alfred
Publication year - 2001
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/1097-0282(20011005)59:4<257::aid-bip1022>3.0.co;2-7
Subject(s) - tropomyosin , chemistry , dimer , coiled coil , heptad repeat , peptide , leucine zipper , crystallography , asparagine , cysteine , monomer , branching (polymer chemistry) , helix (gastropod) , alanine , amino acid , peptide sequence , biochemistry , polymer , organic chemistry , actin , ecology , biology , snail , gene , enzyme
Synthesis and CD and 13 C α ‐NMR studies in a near‐neutral saline buffer are reported for a 65‐residue peptide ( 190 Tm 254 ) comprising residues 190–254 of the α‐tropomyosin chain. CD on a version disulfide cross‐linked via the N‐terminal cysteine side chains indicates that this dimer is highly helical and melts near 48°C. The CD is independent of peptide concentration, showing that association of 190 Tm 254 stops at the two‐strand level. Similar studies on the reduced version show much lower helix content at low temperature, melting points below room temperature, and the expected concentration dependence. The observed melting temperature of the reduced peptide is far below (by 27°C) that expected from an extant analysis of calorimetry data on parent tropomyosin that designates 190 Tm 254 as an independently melting “cooperative block.” This disagreement and the pronounced nonadditivity seen when data for 190 Tm 254 are combined with extant data for other subsequences argue decisively against the concept of specific independently melting blocks within the tropomyosin chain. The data for 190 Tm 254 also serve to test recent ideas on the sequence determinants of structure and stability in coiled coils. Analysis shows that some ideas, such as the stabilizing effect of leucine in the d heptad position, find support, but others—such as the destabilizing effect of alanine in d , the dimer‐disfavoring effect of β‐branching in d and its dimer‐favoring effect in a , and the dimer‐directing effect of asparagine in a —are more questionable in tropomyosin than in the leucine zipper coiled coils. 13 C α ‐NMR data at two labeled sites, L228( d ) and V246( a ), of 190 Tm 254 display well‐separated resonances for folded and unfolded forms at each site, indicating that the transition is slow on the NMR time scale and thus demonstrating the possibility of obtaining thermodynamic and kinetic information on the transition at the residue level. © 2001 John Wiley & Sons, Inc. Biopolymers 59: 257–265, 2001