The structure of a complex between a peptide inhibitor with the sequence N-acetyl-Thr-Ile-Nle-psi[CH2-NH]-Nle-Gln-Arg.amide (Nle, norleucine) with chemically synthesized HIV-1 (human immunodeficiency virus 1) protease was determined at 2.3 A resolution (R factor of 0.176). Despite the symmetric nature of the unliganded enzyme, the asymmetric inhibitor lies in a single orientation and makes extensive interactions at the interface between the two subunits of the homodimeric protein. Compared with the unliganded enzyme, the protein molecule underwent substantial changes, particularly in an extended region corresponding to the "flaps" (residues 35 to 57 in each chain), where backbone movements as large as 7 A are observed.
Alamethicin, a 20-amino acid peptide, has been studied for a number of years as a model for voltage-gated channels. Recently both the x-ray structure of alamethicin in crystal and an NMR solution structure have been published (Fox and Richards, 1982. Bannerjee et al., 1983). Both structures show that the amino end of the molecule forms a stable alpha-helix nine or 10 residues in length and that the COOH-terminal ends exhibits a variable hydrogen bonding pattern. We have used synthetic analogues of alamethicin to test various hypotheses of its mode of action. As a result of these studies we propose a channel structure in which the COOH-terminal residues bond together as a beta-barrel, leaving the alpha- helices free to rotate under the influence of the electric field and gate the channel. Though the number of monomers per channel varies with experimental conditions, the gating charge per monomer stays close to that expected from an alpha-helical gate. We can also alter the sign of the voltage which turns on a channel by varying the charge on the alamethicin analogue. Channels are always slightly cation-selective even though formed by monomers with negative, positive, or zero formal charge. Channels are less stable in low ionic strength solutions than high. Finally, alamethicin conductance parameters vary systematically with changes in membrane thickness. We show how these results and others in the literature can be explained by a fairly detailed structural model. The model can be easily generalized to a form more suited to high molecular weight single-peptide-chain proteins.
Free energy surfaces, or potentials of mean force, for the a-to 3io-helical conformational transition in polypeptides have been calculated in several solvents of different dielectric. The a-to 3io-helical transition has been suggested as potentially important in various biological processes, including protein folding, formation of voltagegated ion channels, kinetics of substrate binding in proteins, and signal transduction mechanisms. This study investigates the thermodynamics of the a-to 3io-helical transition of a model peptide, the capped decamer of a-methylalanine, in order to assess the plausibility of this transition in the mechanisms of such biological processes. The free energy surfaces indicate that in each environment studied the a-helical conformation is the more stable of the two for the decapeptide. The thermodynamic data suggest that the a-helix is energetically stabilized and the 3 io-helix is entropically favored. The inclusion of dichloromethane, acetonitrile, or water results in approximately 7 kcal/mol of relative conformational energy (favoring the a-helix) and 3 kcal/mol of relative conformational entropy (favoring the 3io-helix) in comparison to the gas phase. In polar environments, the a-helix is stabilized by its more favorable solute-solvent electrostatic interactions, and solute-solute steric interactions. In addition, it was concluded that in polar solvents, especially water, it is possible for the peptide to reduce some of the inherent strain of the 3io-helix by widening ip, the resulting weaker intrasolute hydrogen bonds being compensated for by increased hydrogen bonding to the solvent. Lower polarity environments are associated with a marginally increased relative stability of the 3io-helix, which we suggest is largely due to the additional intrahelical hydrogen bond of this conformation. The data suggest that, in environments such as membranes, the interior of proteins or crystals, the complete transition from an a-helix to a 3io-helix for this decapeptide would require less than 6 kcal/mol in free energy. Switching conformations for individual residues is much more facile, and shorter 3io-helices may actually be energetically favored, at least, in nonpolar environments. This study primarily estimates the backbone contribution to the helical transition; side chain interactions would be expected to play a significant role in stabilizing one conformer relative to the other. It is, therefore, quite feasible that the a-to 3io-helical transition could provide a possible mechanism for many biological processes. While there are many factors, such as helix length and side chain packing, that contribute to the selection of either the aor the 3io-helical conformation or a mixture of the two, this study focuses primarily on one of these effects, that of the polarity of the environment.
The precise molecular structure of the antigenic determinant recognized by the T-cell receptor of the CD4-positive cell has not been completely resolved. A major advance in our understanding of this issue has been made by our demonstration of a direct association between an immunogenic peptide and a purified Ia molecule. The most likely and economical hypothesis is that antigen binds directly to an Ia molecule creating the antigenic determinant and that this antigen-Ia complex is recognized by the T-cell receptor. We examined in detail a determinant of hen egg-white lysozyme (HEL) contained in the tryptic fragment HEL(46-61), recognized by T cells in H-2k strains of mice. This peptide binds with a Kd of approximately 3 microM to I-Ak molecules. We have already ascertained that (1) the 10-mer HEL(52-61) is the shortest stimulatory peptide; (2) the Leu56 residue, the only residue different from mouse lysozyme, is responsible for the immunogenicity; (3) the Leu56 and Tyr53 residues are critical for recognition by the T-cell receptor and (4) HEL(46-61) generates multiple determinants when it associated with the I-Ak molecule. If antigen and Ia interact, the antigen must have two features: it must bind to an Ia molecule and also interact with the T-cell receptor. The two sites do not appear to be laterally separable in this peptide and are therefore probably composed of distinct but interspersed amino-acid residues. We have now identified the three residues of HEL(52-61) that contact the T-cell receptor and three other residues that contact the I-Ak molecule. From modelling studies we also propose that HEL(52-61) assumes an alpha-helical conformation as it is bound to I-Ak and recognized by the T-cell receptor.
Due to the difficulties in experimentally differentiating between the a-and 3io-helical conformations in solution, isolated helical peptides have been assumed to be in the -helical conformation. However, recent electron spin resonance (ESR) studies have suggested that such peptides, in particular short alanine-based peptides, are 310helical (Miick, S. M.; et al. Nature 1992, 359, 653-5). This result prompted us to further investigate the helical conformations of alanine-based peptides in solution using electron spin resonance spectroscopy. Unlike previous investigations with a flexible link connecting the spin-label to the peptide backbone, we used a conformationally constrained spin-label (4-amino-4-carboxy-2,2,6,6-tetramethylpiperidine-1 -oxyl, Toac) that is rigidly attached to the peptide backbone. From a combination of molecular modeling and ESR spectroscopy investigations, it was concluded that these alanine-based peptides exist primarily in the -helical conformation, and not the 3io-form as previously suggested for an analogous set of peptides in aqueous environments. This discrepancy is thought to be due to the differences in flexibility of the spin-labels employed. The conformationally constrained spin-label Toac used in this study should accurately reflect the backbone conformation. Free energy surfaces, or potentials of mean force, for the conformational transition of the spin-label used in previous studies (Miick S. M.; et al. Nature 1992, 359, 653-5) suggest that this spin-label is too flexible to accurately distinguish between the a-and 3io-helical conformations.
Assessment of the conformational implications of chemical modification is an important aspect of analogue design. A new procedure, the assessment of conformational mimicry, which determines the percentage of sterically accessible conformations for the parent compound also available to the analogue, is used to show that 88% of the conformers allowed for the cis amide bond are also available to peptides in which the amide bond is replaced by a 1,5-disubstituted tetrazole ring that locks the amide bond in the cis conformer. This analysis was made possible by the crystal structure of a cyclic dipeptide, cyclo[l-Phe-i/'(CN4)-L-Ala], determined in this paper. The crystals of the diketopiperazine analogue are monoclinic, space group P2\/c, with cell parameters a = 11.677 (1), b = 7.742 (1), c = 13.086 (1) A; /? = 93.39 (1)°; Z = 4; and = 1.368 g cm-3. The tetrazole ring system is planar with all five torsional angles equal to 0°. The diketopiperazine ring system is nearly planar, and the phenylalanine ring adopts the flagpole orientation over the cyclic dipeptide. A procedure for the preparation of this class of peptide analogues by synthetic routes avoiding racemization of the amino acids of the starting dipeptide is demonstrated. The tetrazole ring provides, therefore, a synthetic probe for the role of cis-trans isomerism of A'-alkylamide bonds, such as that of proline, in molecular recognition.Replacement of the amide bond by surrogates to enhance metabolic stability and/or probe receptor specificity has become an increasingly important topic of research1 as the central biological role of peptides as chemical effectors becomes more understood. Proline occupies a special role among those amino acids
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.