Observation via one-dimensional 13Cα NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper

Eva G. Lovett, D. André D'Avignon, Marilyn Emerson Holtzer, Emory H. Braswell, Dan Zhu, Alfred Holtzer

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1781-1785
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number5
DOIs
StatePublished - Mar 5 1996
Externally publishedYes

Keywords

  • Coiled coils
  • Protein folding

Fingerprint Dive into the research topics of 'Observation via one-dimensional <sup>13</sup>C<sup>α</sup> NMR of local conformational substates in thermal unfolding equilibria of a synthetic analog of the GCN4 leucine zipper'. Together they form a unique fingerprint.

  • Cite this