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

Abstract

13C alpha 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% 13C alpha 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-coli 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.

Designing Heterodimeric Two-stranded α-Helical Coiled-coils

“…The greatest stability gains were observed at chain lengths of 3 heptads; further increases in length were stabilizing but to lesser degrees. This is primarily because of end fraying, a partial and temporary unfolding of the helix termini (52,82,83). We found that an increase from 3 to 4 heptads caused an increase in stability of 4.06 kcal/mol for the ISAL sequence and an increase of 3.86 kcal/mol for the VAAL sequence.…”

Real-Time Monitoring of the Interactions of Two-Stranded de Novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding

“…This demonstrated that the contribution of Leu-Leu hydrophobic interactions to the stability of the coiled-coil structure were less important at the ends of the coiled-coil, and that the ends of the coiled-coil were more flexible. More recently, Holtzer et al (65) clearly demonstrated by 13 C R chemical shifts that such an end-fraying also occurred in a GCN4-like leucine zipper. All these studies suggest that the different heptads which make up a dimeric coiled-coil structure contribute differently to its stability; the heptads located at the ends of the coiled-coil are able to fray whereas the ones composing the core of the coiled-coil are more stable due to the tighter packing of the hydrophobic residues.…”

Progress towards structural understanding of infectious sheep PrP-amyloid

“…56,57 Thirdly, secondary chemical shifts in unfolded proteins were found to almost be in the range expected for a-helical residues. 58 Whatever the structure of this middle segment is, it is incompatible with the adoption of a uniform in-register b-sheet structure as demonstrated by 1D-solid-state NMR studies on selectively 13 CO-labeled scrapie prion-seeded Syrian hamster recPrP (90-231)-fibrils. 15 (iii) A b-sheet-core C-terminal of »155 is consistent with the finding that Tyr152-Arg154 remains flexible during fibrillation, whereas Tyr165-Arg167 is part of the rigid core.…”