| dc.description.abstract | Proteins and polypeptides play multi?faceted functional roles in the living organism. Numerous biological processes are controlled by these biomolecules. Hence, there is widespread interest in understanding the structure, function, and action of these molecules. Various techniques such as X?ray diffraction, Nuclear Magnetic Resonance (NMR), Circular Dichroism (CD), Infrared (IR), and Raman spectroscopy have been used to elucidate polypeptide and protein conformation. Studies on synthetic model peptides can provide insight into the conformational preferences of specific peptide sequences and the factors influencing backbone folding. This thesis describes attempts aimed at developing acyclic peptides as conformational models. The thesis is divided into eight chapters.
A general introduction to the various regular peptide conformations and their analysis using spectroscopic methods is presented in Chapter I. The importance of introducing conformational constraints using ??aminoisobutyryl (Aib) and/or prolyl residues or disulfide linkages to generate peptide models with reduced conformational flexibility is highlighted.
Chapter II describes the synthesis and spectroscopic studies of model ??turn forming peptides having the general sequence Piv–Pro–X–NHMe, where X = Aib, Gly, D?Ala, L?Ala, L?Val, D?Val, L?Leu, D?Leu, and L?Thr. The ??turn conformations were elucidated using Nuclear Overhauser Effects (NOE), CD, and Raman spectroscopy. NOE experiments provided a basis for differentiating Type II ??turns from Type I and Type III ??turns. Using this technique, Type II ??turns were assigned to the dipeptides with X = Aib, Gly, D?Ala, D?Leu, and D?Val. A Type V ??turn structure was observed for peptides containing L?Ala and L?Leu from NOE studies. Solvent?dependent structural changes were monitored using CD.
Chapter III describes the synthesis and conformational studies on peptides with the sequence Boc?Aib?X?Pro?NHMe, where X = Pro, L?Ala, D?Ala, Gly, Val, and Leu. These systems were examined as possible models for 5?1 hydrogen bonded structures and for evaluating environmental effects on cis–trans isomerism about X–Pro bonds. Low?temperature ¹H NMR studies on Boc?Aib?Pro?Pro?NHMe in CDCl? provide evidence for the presence of multiple conformational states in this peptide. ¹³C NMR studies indicate the presence of cis–trans isomerism in solution, which is sequence and solvent dependent. The all?trans structure is devoid of intramolecular hydrogen bonds, as indicated by solvent? and temperature?dependent NMR studies. X?ray investigations on Boc?Aib?Pro?Pro?NHMe and Boc?Aib?Leu?Pro?NHMe provide supporting evidence for the NMR interpretation. ¹H NMR studies favour a Type VI ??turn in the cis X?Pro conformers.
Chapter IV describes the synthesis and spectroscopic (¹H NMR, CD, and IR) studies on an Aib?containing decapeptide, Boc?Aib?Val?Aib?Aib?Val?Val?Val?Aib?Val?Aib?OMe. This sequence was chosen to evaluate the effect of a triplet of L?amino acids on the conformation of Aib?containing oligopeptides. A brief survey of the conformational preferences of Aib?containing polypeptides is also presented. The results obtained for the decapeptide give evidence for the formation of helical conformers in solution, indicating the strong tendency of Aib residues to dictate helical folding even in the presence of a high content of valine residues. Evidence for structural transitions on going from a non?polar to a polar solvent is provided.
Studies on acyclic peptide models for intramolecular ??sheet conformation are presented in Chapters V and VI. Sequences designed as models for intramolecular ??sheet conformation were of two kinds:
(a) Homovaline peptides having an Aib or Pro residue, or an Aib?Pro segment in the centre of the sequence to nucleate ??turn formation. The sequences synthesized were Boc?(Val)n?X?(Val)m?OMe (n = 2, 3; m = 3; X = Aib, Pro) and Boc?(Val)n?X?Y?(Val)m?OMe (n = 2, 3; m = 3; X?Y = Aib?Pro). ¹H NMR and IR evidence has been provided for the presence of intramolecular ??sheet structures. Solvent?dependent conformational changes have been observed by CD. Evidence is also provided for the population of helical conformations in solvents such as methanol and trimethylphosphate.
(b) Acyclic cystine peptides of the type Boc?Cys??????(Val)n?Trp?OMe (n = 1, 2, 3) were synthesized to explore side?chain interactions in antiparallel ??sheet structures promoted by the disulfide moiety. The quenching of tryptophan fluorescence by the proximal disulfide group was used to identify the conformation of the peptides. Reduction studies using sodium borohydride provide evidence for the formation of antiparallel ??sheet conformation about the S–S bond. These results are presented in Chapter VI.
Synthesis and spectroscopic (NMR and CD) studies on the fragments (Boc?Asp(OBzl)?Leu?Thr?(Gly)??Val?OBzl and Boc?Leu?Thr?(Gly)??Val?OBzl) and analogs (Boc?Asp(OBzl)?Leu?Thr?(Gly)??Val?OBzl and Boc?Leu?Thr?(Gly)??Val?OBzl) of (Thr?)?speract, a sperm?activating peptide, are described in Chapter VII. ¹H NMR evidence for the presence of intramolecular ??sheets, which are highly aggregated in non?polar solvents, is presented. The results indicate that peptides containing a stretch of three glycine residues aggregate to a much greater extent than peptides with two glycine residues.
An appendix to the thesis briefly describes reverse?phase HPIC studies of diastereomeric peptides with the sequence Piv?Pro?X?NHMe, which establish racemization at 1?Pro on activation of pivaloyl?proline.
The main findings of this investigation are summarized in Chapter VIII.
List of Publications:
The type II ??turn conformation of Pivaloyl?L?Prolyl???aminoisobutyryl?Methylamide: Theoretical, Spectroscopic, and X?ray studies. B. V. Venkataram Prasad, Hemalatha Balaram, and P. Balaram. Biopolymers, 21, 1261–1273 (1982).
Multiple conformational states of a Pro?Pro peptide: Structural changes on dissolution of single crystals. Hemalatha Balaram, B. V. Venkataram Prasad, and P. Balaram. Biochem. Biophys. Res. Commun., 109, 825–831 (1982).
Multiple conformational states of a Pro?Pro peptide: Solid state and solution conformations of Boc?Aib?Pro?Pro?NHMe. Hemalatha Balaram, B. V. Venkataram Prasad, and P. Balaram. J. Am. Chem. Soc., 105, 4065 (1983).
Nuclear Overhauser effects and circular dichroism as probes of ??turn conformations in acyclic and cyclic peptides with Pro?X sequences. B. N. N. Rao, A. I. Cumar, H. Balaram, A. Ravi, and P. Balaram. J. Amer. Chem. Soc., 105, 7423–7428 (1983).
Peptide models for ??turns: A circular dichroism study. M. Crisma, G. D. Pasman, H. Balaram, and P. Balaram. Int. J. Pept. Protein Res. (1983).
Acyclic peptides as conformational models: Crystal structure of Boc?Aib?Leu?Pro?NHMe·2H?O. B. V. V. Prasad, H. Balaram, and P. Balaram. Int. J. Pept. Protein Res. (1984).
Side?chain interactions as probes of ??sheet structures in peptides: Fluorescence quenching of tryptophan by proximal cystine residues. Hemalatha Balaram and P. Balaram (Submitted).
Acyclic oligopeptide models for ??turns, ??sheets, and helical structures. Hemalatha Balaram, A. Ravi, E. K. S. Vijayakumar, and P. Balaram. Presented at the Eighth American Peptide Symposium, Tucson, Arizona, U.S.A., May 22–27 (1983). | |
