TY - JOUR
T1 - Observation via one-dimensional 13Cα NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper
AU - Lovett, Eva G.
AU - D'Avignon, D. André
AU - Holtzer, Marilyn Emerson
AU - Braswell, Emory H.
AU - Zhu, Dan
AU - Holtzer, Alfred
PY - 1996/3/5
Y1 - 1996/3/5
N2 - Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Cα at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73°C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (>>2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is ≥10-20 ms.
AB - Synthesis of a 33-residue, capped leucine zipper analogous to that in GCN4 is reported. Histidine and arginine residues are mutated to lysine to reduce the unfolding temperature. CD and ultracentrifugation studies indicate that the molecule is a two-stranded coiled coil under benign conditions. Versions of the same peptide are made with 99% 13Cα at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal unfolding equilibria over the entire transition from 8 to 73°C. Spectra at the temperature extremes establish the approximate chemical shifts for folded and unfolded forms at each labeled site. Resonances for each amino acid appear at both locations at intermediate T, indicating that folded and unfolded forms interconvert slowly (>>2 ms) on the NMR time scale. Moreover, near room temperature, the structured form's resonance is double at several, but not all, sites, indicating at least two slowly interconverting, structured, local conformational substates. Analysis of the dynamics is possible. For example, near room temperature at the Val-9, Ala-24, and Gly-31 positions, the equilibrium constant for interconversion of the two structured forms is near unity and the time scale is ≥10-20 ms.
KW - Coiled coils
KW - Protein folding
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U2 - 10.1073/pnas.93.5.1781
DO - 10.1073/pnas.93.5.1781
M3 - Article
C2 - 8700835
AN - SCOPUS:0029987133
SN - 0027-8424
VL - 93
SP - 1781
EP - 1785
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 5
ER -