Current ideas on unfolding equilibria in two-chain, coiled-coil proteins are examined by studies of a species of beta beta tropomyosin that is sulfhydryl blocked at C190 and disulfide cross-linked at C36 (.beta-beta.). T...Current ideas on unfolding equilibria in two-chain, coiled-coil proteins are examined by studies of a species of beta beta tropomyosin that is sulfhydryl blocked at C190 and disulfide cross-linked at C36 (.beta-beta.). The desired species is produced by a seven-step process: (1) Rabbit skeletal muscle, comprising predominantly alpha alpha and alpha beta species, is oxidized with ferricyanide, cross-linking both species at C190. (2) The product is carbamylated at C36 of beta chains, using cyanate in denaturing medium at pH 6. (3) All C190 cross-links are reduced with dithiothreitol (DTT). (4) All C190 sulfhydryls are permanently blocked by carboxyamidomethylation. (5) Chromatography on carboxymethylcellulose in denaturing medium is used to separate C190-blocked alpha chains from C190-blocked, C36-carbamylated beta chains. (6) The latter are decarbamylated in denaturing medium by raising the pH to 8.0. (7) The C190-blocked beta chains are renatured and cross-linked at C36 by ferricyanide. The procedure and the quality of the final product are judged by NaDodSO4/polyacrylamide gel electrophoresis, titration of free sulfhydryls, and electrophoretic analysis of trypsin digestion products. Thermal unfolding curves are reported for the resulting pure .beta-beta. species and for its DTT-reduction product. The latter (.beta beta.) show equilibrium thermal unfolding curves that are very similar to those of the parent beta beta noncross-linked species. The .beta-beta. cross-linked species unfolds in a single-phase, cooperative transition with a melting temperature intermediate between the pretransition and posttransition shown by its cross-linked counterpart, the C190 cross-linked, C36-blocked species (.beta-beta.), which was studied earlier. These transitions are compared with one another and with that of the doubly cross-linked species, beta-(-)beta, in the light of two extant physical models for such transitions. The all-or-none segments model successfully rationalizes the data qualitatively for the .beta-beta. and .beta-beta. species if the usual postulates of greater inherent stability of the amino vs the carboxyl end of the molecule and of strain at each cross-link are accepted. However, the same model then requires that the beta-(-)beta species be the least stable of the three, whereas experiment shows the opposite, thus falsifying the all-or-none segments model. The continuum-of-states model is also qualitatively in accord with data on the .beta-beta. and .beta-beta. species.(ABSTRACT TRUNCATED AT 400 WORDS)
Solvent effects on the thermodynamics of two processes--folding of proteins and association between proteins--are examined in detail. A complete inventory of the multitude of solvent effects may be obtained by employing...Solvent effects on the thermodynamics of two processes--folding of proteins and association between proteins--are examined in detail. A complete inventory of the multitude of solvent effects may be obtained by employing the concept of conditional solvation free energy. This theoretical tool allows for the isolation of specific side-chain effects from the entire protein and for the study of its contribution to the overall free energy change in small model compounds. Some numerical examples are presented, and ways of estimating other cases, for which no relevant experimental data are available, are suggested. Our findings lead to the conclusion that the currently used hydrophobicity scales, based on partition coefficients between water and an organic solvent, are inadequate measures of the contribution of side chains being transferred from water to the interior of the protein. We have also tentatively concluded that correlation between hydrophilic functional groups might be more important than correlations between hydrophobic side chains.
The CH-stretching vibrational CD (VCD) spectra of glycyl-L-alanine, L-alanylglycine, and L-alanyl-L-alanine have been studied at neutral, high, and low pH in D2O solution. The intense positive VCD band attributed to the...The CH-stretching vibrational CD (VCD) spectra of glycyl-L-alanine, L-alanylglycine, and L-alanyl-L-alanine have been studied at neutral, high, and low pH in D2O solution. The intense positive VCD band attributed to the C alpha H stretch of the alanyl residue in glycyl-L-alanine at neutral pH is absent in L-alanylglycine. In contrast to the VCD spectra of L-alanine, the positive methine-stretching VCD band in glycyl-L-alanine and L-alanyl-L-alanine is still present at pH 2. Based on the ring current mechanism, the VCD spectra are consistent with the presence of a five-membered CO...HN intramolecular hydrogen-bonded ring between the C-terminal carboxylate and peptide NH groups at neutral and high pH; and a seven-membered COH...O = C hydrogen-bonded ring between the C-terminal carboxyl OH and peptide C = O groups at low pH. In the N-terminal alanyl residue, the peptide C = O group is hydrogen bonded to the NH trans to the methine bond. The CH-stretching VCD spectra of L-alanyl-L-alanyl-L-alanine at neutral pH are consistent with two intramolecularly hydrogen-bonded conformations for the central alanyl residue.
The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were s...The synthetic peptide of sequence H-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-OH, termed peptide T, a competitor of the Human Immunodeficiency Virus in the binding to human T cells, and its C-terminal pentapeptide fragment, were studied by 1H-nmr in DMSO solution to determine conformational preferences. The observation of nuclear Overhauser enhancements (NOEs) for both peptides, and unusual finding for small linear peptides, allowed complete sequence-specific resonance assignments. Long-range NOEs, ring-current shifts, and the very small temperature coefficient of the Thr8 NH chemical shift suggest, for the zwitterionic form of peptide T, the presence in solution of a beta-turn involving Thr5, Asn6, Tyr7 and Thr8. This conformational feature is consistent with previous structure-activity relationship studies indicating the invariance of the same residues in several potent pentapeptide analogues. The studied pentapeptide fragment, although less structured, shows some tendency to fold even in a polar solvent such as DMSO. Preliminary chemotaxis data on some pentapeptide analogues are consistent with our structural model.
The conformation of the tridecapeptide alpha-factor of the yeast Saccharomyces cerevisiae was examined in both solution and in the presence of lipid vesicles. CD, differential scanning calorimetry, and phosphorus nmr all...The conformation of the tridecapeptide alpha-factor of the yeast Saccharomyces cerevisiae was examined in both solution and in the presence of lipid vesicles. CD, differential scanning calorimetry, and phosphorus nmr all indicate that this mating pheromone interacts with lipid vesicles. In both aqueous and organic solution the alpha-factor is a flexible molecule that exhibits features of a type II beta-turn spanning the center of the peptide. Two-dimensional Nuclear Overhauser enhancement spectroscopy gives evidence that the beta-turn is stabilized on interaction of the peptide with lipid vesicles. Our current belief is that the beta-turn may play an important role in the biologically active conformation of the alpha-factor.
The major immunodominant region of the coating protein of Plasmodium falciparum sporozoites contains multiple tandem copies of the sequence Asn-Ala-Asn-Pro (NANP). Current efforts for the development of an antisporozoite...The major immunodominant region of the coating protein of Plasmodium falciparum sporozoites contains multiple tandem copies of the sequence Asn-Ala-Asn-Pro (NANP). Current efforts for the development of an antisporozoite vaccine are focused on the synthesis of polypeptides reproducing part of the circumsporozoite protein repeat sequence and, in an attempt to relate conformational properties and biological response, 1H-nmr one- and two-dimensional studies of the synthetic models (NANP)2NA and (NANP)6 were carried out in water and water/methanol mixtures, at 400 and 500 MHz. In water, (NANP)6 undergoes fast conformational averaging. At variance, in water/methanol, the molecule appears to adopt an extensive structure, but detailed analysis is impaired by high spectral degeneracy. Based on the results obtained with (NANP)2NA and from preliminary experiments in water/trifluoroethanol, an interpretation is suggested for the (NANP)6 data in water/methanol in terms of a mixed sequence of beta I-turns and half-turns (or/and gamma I-turns) around the positions Ni-1-Pi-Ni + 1.