Collagen-like peptides with potential for ion pair formation were studied to investigate the role of electrostatic interactions in the triple-helix conformation. Three peptides--(POG)10, the EK-containing peptide (POG)4EKG(POG)5, and T3-487, a peptide with 18 residues of type III collagen and a C-terminal (GPO)4 tail--all form stable triple helices in aqueous solution, with melting temperatures of 58, 46, and 26 degrees C, respectively, at neutral pH. The thermal stabilities of these peptides correlate with their imino acid content, which is 66%, 60%, and 41%, respectively. Variation of pH over the range of 1-13 led to 8-9 degrees C changes in the Tm of the EK-containing peptide and peptide T3-487, with the greatest stability seen at pH values where both acidic and basic residues are ionized. Equilibrium ultracentrifugation shows these peptides are largely trimeric at low temperature, with no hexamers or larger aggregates, indicating that the pH-dependent stability arises from intramolecular interaction. Computer modeling indicates both intrachain ion pairs and interchain ion pairs can form and stabilize the triple helix. Studies of the pH dependence of the thermal stability of (POG)10 and the N-terminal acetylated form of T3-487 indicate that repulsion of the three charged N-terminal or C-terminal ends has a destabilizing effect. Taking into account these end effects, the energy contribution of two oppositely charged residues in a triple helix which are sterically capable of participating in ion pairs and backbone hydrogen bonding is 0.5-1 kcal/mol ion pair. It is possible that the stabilizing influence of ion pairs arises indirectly, through elimination of like charge repulsion, formation of ion pairs in the single chain form, or solvent effects.

Glycine is found as every third residue along the entire length of triple helices in fibrillar collagens, but the triple-helix regions of nonfibrillar collagens and other proteins usually contain one or more interruptions in this repeating pattern. A set of four peptides was designed to model the effect of interruptions in the (Gly-X-Y)n repeating pattern on triple-helix formation, stability, and folding. Into the middle of the stable triple-helical peptide (Pro-Hyp-Gly)10, an interruption was introduced representing one of the four possible categories: a glycine deletion, a deletion of a hydroxyproline (Y position), an alanine insertion, or a glycine to alanine substitution. As shown by sedimentation equilibrium, NMR, and CD studies, the introduction of an interruption still allowed formation of trimers in solution, but with marked decrease in stability. The degree of destabilization and the thermodynamic basis for the loss of stability depended on the type of interruption. The glycine substitution and alanine insertion were the least disruptive, followed by the hydroxyproline deletion, with the glycine deletion being the most destabilizing. Our results suggest that the breaks in these peptides affect both the triple-helical conformation and the monomer conformation. These studies provide a basis for considering the structural and functional consequences of different kinds of interruptions in collagen.

Trimerization in Aqueous Solutions of Methylene Blue 2477 motic coefficients4 can be used to predict the variation of «12 and «21 with ionic strength if S' = 0.190 (obtained at I > 3) is assumed to be independent of ionic strength, (c) The best values of the Earned rule coefficients in the ionic strength range from 1 to 6 are « 2 = 0.048 ± 0.003 and «21 = -0.035 ± 0.005, essentially independent of ionic strength. There is no clear reason for the discrepancy between our measurements and those of Lanier, but we believe our values of Earned rule coefficients are more likely to be correct, since they have been obtained by two independent experiments using both chloride-reversible and sulfate-reversible electrodes, and are also consistent with a thermodynamically based extrap-olation from higher ionic strengths, where our results are in agreement with those of Lanier.The behavior of the Earned rule coefficients for this system at ionic strengths below 0.5 is not yet established. This is of theoretical rather than practical interest, since in this range even large deviations from the above values will not introduce appreciable error in calculated activity coefficients.

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Though many proteins in the cell are large and multimeric, their folding has not been extensively studied. We have chosen SecA as a folding model because it is a large, homodimeric protein (monomer molecular mass of 102 kDa) with multiple folding domains. SecA is the ATPase for the Sec-dependent preprotein translocase of many bacteria. SecA is a soluble protein that can penetrate into the membrane during preprotein translocation. Because SecA may partially unfold prior to its insertion into the membrane, studies of its stability and folding pathway are important for understanding how it functions in vivo. Kinetic folding transitions in the presence of urea were monitored using circular dichroism and tryptophan fluorescence, while equilibrium folding transitions were monitored using the same techniques as well as a fluorescent ATP analogue. The reversible equilibrium folding transition exhibited a plateau, indicating the presence of an intermediate. Based on the data presented here, we propose a three-state model, N(2) if I(2) if 2U, where the native protein unfolds to a dimeric intermediate which then dissociates into two unfolded monomers. The SecA dimer was determined to have an overall stability (DeltaG) of -22.5 kcal/mol. We also investigated the stability of SecA using analytical ultracentrifugation equilibrium and velocity sedimentation, which again indicated that native or refolded SecA was a stable dimer. The rate-limiting step in the folding pathway was conversion of the dimeric intermediate to the native dimer. Unfolding of native, dimeric SecA was slow with a relaxation time in H(2)O of 3.3 x 10(4) s. Since SecA is a stable dimer, dissociation to monomeric subunits during translocation is unlikely.

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...

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