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.

A method is presented for determining the concentrations of peptides and proteins having isodichroic points near 203 nm. The existence of an isodichroic point for a given substance indicates a local two-state (alpha-helix, random coil) population. The mean residue ellipticity at the isodichroic point, [theta lambda i], is, of course, independent of helix content. For a wide variety of synthetic and natural peptides, including both single helices and coiled coils, it is shown that [theta lambda i] is also essentially independent of substance and of whether the transition is induced by temperature, ionic strength, pH, chain length changes, amino acid substitution, or solvent perturbation. Averaging [theta lambda i] values culled from various laboratories gives -151 +/- 16 (SD, 7 sources) deg.cm2.mmol-1. In our laboratory, nonpolymerizable rabbit alpha-tropomyosin and two alpha-tropomyosin subsequences yield -135 +/- 10 (SD, 190 values) deg.cm2.mmol-1. Thus, given [theta lambda i] for a peptide of known concentration, it is possible to estimate the concentration of any other peptide provided that it has an isodichroic point at which the ellipticity is accurately measurable. It is then possible to calculate [theta lambda] at any other wavelength for which theta is known. It is advisable to determine [theta lambda i] for the best known peptide in one's own laboratory, since it depends on absolute instrument and cell calibrations and an absolute concentration determination.

New data are presented for the a-helix content of non-cross-linked and cross-linked a-tropomyosin at near-neutral pH as a function of temperature (20-70 "C) and over a 1000-fold range of protein concentration. Helix content is obtained from circular dichroism measurements by a new method. Experimental thermal denaturation curves are dependent upon concentration for the non-cross-linked protein but independent of it for the cross-linked protein, indicating the mass actional effect of chain dissociation in the non-cross-linked case. When the data are used to determine the helix-helix interaction parameter [w(Z')] introduced earlier and the resulting w(T) is used in the realized theory previously developed, a rather satisfactory fit of theory to experiment is obtained over the entire investigated range of temperature and protein concentration. The theory also generates predictions concerning the average molecular weight as a function of temperature for the non-cross-linked protein at a given concentration. The w ( T ) obtained for cy-tropomyosin yields values for various standard thermodynamic properties (AG", AH", AS", and AC,") characterizing the helix-helix interaction. Comparison is made with those obtained earlier from extant data on a short-chain synthetic model coiled coil (XY,) whose hydrophobic helix-helix interactions are entirely due to leucine-leucine contacts. The interaction free energy in XYs is seen to be more favorable than in a-tropomyosin, largely because of its enthalpic advantage; the standard entropy of the interaction actually favors a-tropomyosin. The percent helix calculated from theory for cross-linked chains is always larger than that obtained by experiment. This disagreement is ascribed to distortions in local short-range [a and 4271 and/or long-range [w(Z')] interactions attendant upon formation of the disulfide link-effects not encompassed by the present theory or reflected in the parameters used to realize it. The theory is thus shown to provide a rational framework that serves to separate out effects of mass action and local distortions in comparing non-cross-linked and cross-linked proteins, since it has previously been thought that cross-linking destabilizes only part of the molecule and stabilizes the rest. Similarly, the theory is shown to allow the effects of differences in chain length, cross-linking, and helix-helix interaction to be clearly distinguished when a-tropomyosin is compared with the smaller, synthetic model coiled coil XY6.

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...

A 33-residue pseudo-wild-type GCN4 leucine zipper peptide is used to probe the equilibrium conformational population in proteins. 13Calpha-NMR shows that chain sites differ in structural content at a given temperature, and that two dimeric folded forms are evident at many sites. Spin inversion transfer experiments are reported bearing on the thermodynamics and kinetics of interconversion of the two dimeric folded forms (Fa <--> Fb) at the 13Calpha-labeled position L13. At each temperature, at conditions wherein the population of unfolded chains is quite small, inversion of the Fa spins via a tuned Gaussian pi-pulse is followed by a time interval (tau), interrogation, and recording of the free induction decay. Fifteen such inversions, with varying tau, provide the time course for recovery of equilibrium magnetization after inversion. Similar experiments follow inversion of the Fb spins. Re-equilibration is known to be modulated by four first-order rate constants: two (T1a(-1) and T1b(-1)) for spin-lattice relaxation intrinsic to the respective sites, and two (kab and kba) for the conformational change. All four follow from joint, Bayesian analysis of all the data at each temperature. The equilibrium constant at each temperature for this local transition, determined simply from the equilibrium relative magnetizations at Fa and Fb sites, agrees well with the kinetic ratio kab/kba. The standard Gibbs energies, enthalpy, and entropy follow. Activation parameters, both ways, are accessible from the rate constants and suggest a transition state with high Gibbs energy and enthalpy, but with entropy between those of Fa and Fb.