Thermal unfolding in a GCN4-like leucine zipper: 13C(α) NMR chemical shifts and local unfolding curves

Marilyn Emerson Holtzer, Eva G. Lovett, D. André D'Avignon, Alfred Holtzer

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Abstract

13Cα chemical shifts and site-specific unfolding curves are reported for 12 sites on a 33-residue, GCN4-like leucine zipper peptide (GCN4-lzK), ranging over most of the chain and sampling most heptad positions. Data were derived from NMR spectra of nine synthetic, isosequential peptides bearing 99% 13Cα at sites selected to avoid spectral overlap in each peptide. At each site, separate resonances appear for unfolded and folded forms, and most sites show resonances for two folded forms near room temperature. The observed chemical shifts suggest that 1) urea-unfolded GCN4-lzK chains are randomly coiled; 2) thermally unfolded chains include significant transient structure, except at the ends; 3) the coiled-coil structure in the folded chains is atypical near the C-terminus; 4) only those interior sites surrounded by canonical interchain salt bridges fail to show two folded forms. Local unfolding curves, obtained from integrated resonance intensities, show that 1) sites differ in structure content and in melting temperature, so the equilibrium population must comprise more than two molecular conformations; 2) there is significant end-fraying, even at the lowest temperatures, but thermal unfolding is not a progressive unwinding from the ends; 3) residues 9-16 are in the lowest melting region; 4) heptad position does not dictate stability; 5) significant unfolding occurs below room temperature, so the shallow, linear decline in backbone CD seen there has conformational significance. It seems that only a relatively complex array of conformational states could underlie these findings.

Original languageEnglish (US)
Pages (from-to)1031-1041
Number of pages11
JournalBiophysical journal
Volume73
Issue number2
DOIs
StatePublished - Aug 1997

Bibliographical note

Funding Information:
We acknowledge important discussions with Dr. G. Larry Bretthorst con- cerning the integration of NMR data by Bayesian analysis. This work was supported in part by grant GM 20064 from the Division of General Medical Sciences, U.S. Public Health Service, and a grant from the Muscular Dystrophy Association.

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