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

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% 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...

Site‐specific experiments on folding/unfolding of Jun coiled coils: Thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine‐18

“…13 The 99% 13 C alabeled 9-fluorenylmethoxycarbonyl (Fmoc) leucine was also prepared as in prior work. 18 Analytical high performance liquid chromatography (HPLC) was used after each NMR and CD experiment to check for sample damage.…”

“…In the case of GCN4-like leucine zippers, for example, there is ample evidence that neither the dimer nor the monomer ensemble is conformationally uniform. [16][17][18]30,31 The Jun-lz dimer may contain transient unfolded regions and the monomer transient folded regions that are temperature sensitive. Moreover, either or both may ''cold denature,'' leading to individual unfolding curves that may not be monotonic.…”

“…Further insight can doubtless be gained into the molecular nature of the dimer and monomer ensembles by study of peptides with 13 C a labels at other sites, particularly those distant from the center of the chain, as has been reported for the GCN4-lzK peptide. 18,30 Indeed, such studies have begun here, but are at a preliminary stage.…”

CD and13C?-NMR studies of folding equilibria in a two-stranded coiled coil formed by residues 190-254 of ?-tropomyosin

“…20 These were introduced at the L39,228(d) and V57,246(a) positions, respectively, for samples to be used for NMR experiments. We specify the labeled sites by giving the amino acid type followed by its number in the synthetic peptide's sequence followed by its number in the 284-residue ␣-tropomyosin sequence, followed by parenthetical expression of its heptad position.…”

“…20,26 Apparently, it is slow on the NMR time scale. If the rate were rapid, only one broadened resonance would appear at each residue site.…”