No 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.

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% 13C" at selected sites. One-dimensional 13C NMR spectra are assigned by inspection and used to study thermal Protein conformational equilibria are of vital interest in biochemistry and biophysics and are under intense scrutiny in many laboratories. In this endeavor, studies of coiled-coil proteins have attracted increasing attention, both because of the variety of proteins in which the motif occurs and because its structural simplicity recommends it as a model. A twostranded coiled coil, in which two right-handed a-helices are arranged in parallel and in register and with a slight lefthanded super twist, exhibits all the secondary and higher order structural interactions that are so characteristic of proteins but in a simple repetitive geometric context (1). The structure results from a sequential pattern, called a pseudo-repeating heptad (designated abcdefg), in which residues a and d are hydrophobic and e and g are oppositely charged (1).A physical understanding of protein conformational equilibria requires elucidation of the population of molecular states. Long coiled coils, such as the tropomyosins from rabbit muscle, show multistate unfolding equilibria whose details are controversial (2, 3). Other tropomyosins (e.g., from chicken gizzard) are thought by some (4) to unfold all-or-none (i.e., to comprise only two kinds of molecular conformations: intact two-stranded coiled coils and random monomer chains), but this too is disputed (5).For smaller coiled coils, such as the GCN4 leucine zipper (6), calorimetry evidence fits all-or-none unfolding (7). However, an NMR study of 1H-2H exchange at the amide protonsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.shows that individual residues vary in protection over a million fold range (8). Such differences in local dynamics and stability strongly suggest that substates exist. Finally, although the x-ray crystal structure of the GCN4 leucine zipper shows that the two chains are conformationally distinct (9), extant twodimensional NMR shows only one resonance per proton, indicating that the two chains either become equivalent in solution or rapidly interconvert (10).Evidence concerning such substates can only come from techniques, such as NMR, that are site-specific and are applied to models small enough to ensure interpretability of the data. To that end, we employ a synthetic, 33-residue capped leucine zipper, GCN4-lzK, which is like that of GCN4 except for four conservative mutations: R1K, H18K, R25K, and R33K. That is, in GCN4-lzK, all basic residues are lys...

No abstract