CD and 13Cα-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of α-tropomyosin

Marilyn Emerson Holtzer, Lisa Mints, Ruth Hogue Angeletti, D. André D'Avignon, Alfred Holtzer

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Synthesis and CD and 13Cα-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide (190Tm254) 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 190Tm254 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 190Tm254 as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for 190Tm254 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 190Tm254 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. 13Cα-NMR data at two labeled sites, L228(d) and V246(a), of 190Tm254 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.

Original languageEnglish (US)
Pages (from-to)257-265
Number of pages9
JournalBiopolymers
Volume59
Issue number4
DOIs
StatePublished - Oct 5 2001

Fingerprint

Tropomyosin
Dimers
Nuclear magnetic resonance
Peptides
Freezing
Melting point
Melting
Leucine Zippers
Calorimetry
Temperature
Transition Temperature
Fasteners
Asparagine
Thermodynamics
Leucine
Disulfides
Alanine
Cysteine
Buffers
Display devices

Keywords

  • Leucine zipper
  • Protein denaturation
  • Protein folding

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biophysics

Cite this

CD and 13Cα-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of α-tropomyosin. / Holtzer, Marilyn Emerson; Mints, Lisa; Angeletti, Ruth Hogue; D'Avignon, D. André; Holtzer, Alfred.

In: Biopolymers, Vol. 59, No. 4, 05.10.2001, p. 257-265.

Research output: Contribution to journalArticle

Holtzer, Marilyn Emerson ; Mints, Lisa ; Angeletti, Ruth Hogue ; D'Avignon, D. André ; Holtzer, Alfred. / CD and 13Cα-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of α-tropomyosin. In: Biopolymers. 2001 ; Vol. 59, No. 4. pp. 257-265.
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abstract = "Synthesis and CD and 13Cα-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide (190Tm254) 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 190Tm254 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 190Tm254 as an independently melting {"}cooperative block.{"} This disagreement and the pronounced nonadditivity seen when data for 190Tm254 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 190Tm254 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. 13Cα-NMR data at two labeled sites, L228(d) and V246(a), of 190Tm254 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.",
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T1 - CD and 13Cα-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of α-tropomyosin

AU - Holtzer, Marilyn Emerson

AU - Mints, Lisa

AU - Angeletti, Ruth Hogue

AU - D'Avignon, D. André

AU - Holtzer, Alfred

PY - 2001/10/5

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N2 - Synthesis and CD and 13Cα-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide (190Tm254) 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 190Tm254 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 190Tm254 as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for 190Tm254 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 190Tm254 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. 13Cα-NMR data at two labeled sites, L228(d) and V246(a), of 190Tm254 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.

AB - Synthesis and CD and 13Cα-NMR studies in a near-neutral saline buffer are reported for a 65-residue peptide (190Tm254) 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 190Tm254 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 190Tm254 as an independently melting "cooperative block." This disagreement and the pronounced nonadditivity seen when data for 190Tm254 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 190Tm254 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. 13Cα-NMR data at two labeled sites, L228(d) and V246(a), of 190Tm254 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.

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