Design of complex protein folds requires complete understanding of the stereochemical principles that govern polypeptide chain folding. Extensive studies on design and synthesis of specific secondary structures like β-helices, β -sheets and hairpins have taught us that the unnatural amino acid aminoisobutyric acid (Aib) can be successfully employed for helix nucleation and tight turns of appropriate stereochemistry are facilitated by the use of DPro-Xxx sequences. Availability of such rigid secondary structure scaffolds therefore permits the design of synthetic peptides that can be used as models for investigation of tertiary interactions, primarily that of aromatic residues. Chapter 1 summarizes the present knowledge of peptide design using non-protein amino acids. The chapter also details the unique features of aromatic amino acids, especially tryptophan, and their employment as secondary structure stabilizing elements. Chapters 2-7 contain detailed descriptions of the work carried out on design, synthesis, and structural characterization of designed peptides containing aromatic amino acids. In Chapter 2, the use of aromatic pairs in strand segments of peptide hairpins has been discussed with the results clearly indicating that aromatic interactions at the non-hydrogen bonding position of peptide hairpins contribute to structure stability. In Chapter 3, accommodation of the Leu-Trp-Val segment in helical scaffolds the role of Trp residues in crystallization has been discussed. Chapter 4 outlines the influence of a large number of Trp residues on the preferred backbone conformation, with the studies clearly indicating a preference for helical scaffolds in small peptides. The role of Trp residues at turn regions of peptide hairpins has been discussed in Chapter 5, using examples from both synthetic peptides and from natural peptides containing Pro-Trp segments. The studies suggest that the Pro-Trp segments serve as helix nucleators and disrupt formation of peptide hairpins. The results of this study have been further extended to Conus monile peptides, discussed in Chapter 6. The studies also suggest the role of an aromatic-Pro segment on the cis-trans isomerization of the Xxx-Pro tertiary amide unit. Chapter 7 discusses the contribution of a Cys-His vs Tyr-His pair on strand segment stability in diproline nucleated peptide hairpins. Chapter 8 summarizes the key findings of the work. Chapter 9 lists the references cited in the thesis and the Appendix chapter provides details of experimental techniques used in the study.β

This thesis set out to explore the conformational properties of short designed peptide sequences, in which transitions between structural states may be anticipated. The use of conformationally constrained residues like α-aminoisobutyric acid (Aib) and D-proline (DPro) permits the design of model sequences for structural studies. The principle of imposing conformational constraints by multiple substitutions at backbone atoms in aminoacid residues may also be extended to the higher homologs of α-amino acids, namely β and residues. The experimental results presented in this thesis also examine the potential of using cross-strand interactions between aromatic residues as a probe of structure in designed peptide β-hairpins. Chapter 1 provides a very brief introduction to the necessary background on which the experimental studies in this thesis are based. Chapter 2 describes studies aimed at establishing chain length effects on helix-hairpin conformational distributions in short synthetic sequences, containing centrally positioned Aib-DAla and Aib-Aib segments.The Aib-DAla dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7) and Boc-Aib-Xxx-NHMe (4, 8), where Xxx = DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7) and Boc-Aib-Aib-NHMe (8) local helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3OH. β-Turn conformations (type-I /III) stabilized by intramolecular 4 1 hydrogen bonds are observed for the peptide Boc-Aib-DAla-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 310-helical conformation stabilized by three 4 1 hydrogen bonds. The peptide Boc-Val-Aib-DAla- Leu-NHMe (3) adopts a novel -turn conformation, stabilized by three intramolecular hydrogen bonds (two 4 1 and one 5 1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of the NOE Val(1) NH HNCH3 (5), in CD3OH, suggests that the solid state conformation of peptide 3 is maintained in methanol solutions. Peptide hairpins provide an ideal scaffold for exploring cross-strand interactions between residues on facing antiparallel strands. Chapter 3 reports studies directed towards probing, aromatic interactions between facing Phe residues, positioned at the non-hydrogen bonding positions in designed octapeptide β-hairpins. The studies described in this Chapter employ ring current shifted aromatic proton resonances as a means of probing aromatic ring orientations. Crystal structures of eight peptide -hairpins with the sequence Boc-Leu-Phe-Val-Xxx-Yyy-Leu-Phe-Val-OMe revealed that the Phe(2) and Phe(7) aromatic rings are in close spatial proximity, with a centroid-centroid distance (Rcen) of 4.4Å to 5.4Å between the two phenyl rings. Proton NMR spectra in chloroform and methanol solutions reveal a significant upfield shift of the Phe(7) C , ′ H2 protons (6.65 ppm to 7.04 ppm). Specific assignments of the aromatic protons have been carried out in the peptide Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe (6). The anticipated ring current shifts have been estimated from the aromatic ring geometries observed in crystals for all eight peptides. Only one of the C , ′ H proton lies in the shielding zone, with rapid ring flipping, resulting in averaging between the two extreme chemical shifts. An approximate estimate of the population of conformations which resemble crystal state orientations may be obtained. Key nuclear Overhauser effects (NOEs) between facing Phe sidechains provide support for close similarity between the solid state and solution conformations. Temperature dependence of aromatic ring proton chemical shifts and line widths for peptide 6 (Boc-Leu-Phe-Val-DPro-LPro-Leu-Phe-Val-OMe) and the control peptide Boc-Leu-Val-Val-DPro-Gly-Leu-Phe-Val-OMe establish an enhanced barrier to ring flipping, when the two Phe rings are in proximity. Modeling studies suggest that small, conformational adjustments about the C -C ( 1), and C -C ( 2) bonds of the Phe residues may be required in order to permit unhindered, uncorrelated flipping of both the Phe rings. The maintenance of specific aromatic ring orientations in organic solvents provides evidence for significant stabilizing interactions. Earlier studies from this laboratory established that a centrally positioned DPro-LPro-DAla segment could induce hairpin formation in nonapeptide sequences, facilitated by a three residue loop segment. The DAla residue at position 6 in the nonapeptide Boc-Leu-Phe-Val-DPro-LPro-DAla-Leu-Phe-Val-OMe has been shown to adopt a left handed helical (αL) conformation. The studies described in Chapter 4, examine the effects of aminoacid replacements at positions 5 and 6. NMR studies on eight nonapeptides, with the general sequence Boc-Leu-Phe-Val-DPro-Xxx-Yyy-Leu-Phe-Val-OMe are described. In the case of peptides with a central DPro-LPro-Yyy sequence, two kinds of hairpin conformations are formed in solution. These are; i) β-hairpin structures with a central three residue loop, resulting in registered antiparallel tripeptide strands, and ii) a slipped hairpin structure, nucleated by a central DPro-LPro type-II β-turn, with residue 6 being incorporated into the C-terminal strand. The three residue loop β-hairpins are favored for DAla(6) and Aib(6), while the LAla(6) peptide favors a “slipped” hairpin structure. Replacement of the Pro(5) residue by LAla results in a reduced population of three residue hairpins in the nonapeptide with the DPro-LAla-DAla segment. Replacement of Pro(5) by Aib, abolished hairpin formation. Aromatic proton chemical shifts provide a convenient diagnostic for the presence of three residue loop hairpin conformations in these nonapeptides. A great deal of current interest has focused on the conformations of peptides incorporating β and γ aminoacid residues. Earlier studies from this laboratory have focused on the conformational properties of the β,β -disubstituted γ residue gabapentin (1-aminomethylcyclohexane acetic acid). Subsequent work with the related β aminoacid β3,3Ac6c (1-aminocyclohexaneacetic acid) revealed that intramolecularly hydrogen bonded conformations are infrequently observed in short peptides. The studies described in Chapter 5, examine the conformational properties for model peptides containing the isomeric β-aminoacid, β2,2Ac6c (1-aminomethylcyclohexane-1-carboxylic acid). The effect of gem dialkyl substituents on the backbone conformations of amino acid residues in peptides, has been investigated using four model peptides, Boc-Xxx-2,2Ac6c-NHMe [Xxx = Leu (1), Phe(2)] and Boc-Xxx- 3,3Ac6c-NHMe [Xxx = Leu (3), Phe(4)]. Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed a C11 helical turn, which is a backbone expanded analog of the type III -turn in sequences. The crystal structure of the peptide Boc-Phe- 3,3Ac6c-NHMe (4) establishes a the asymmetric unit adopt backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopts an unfavourable backbone conformation, with the energetic penalty being offset by favourable aromatic interactions between proximal molecules in the crystal. NMR studies provide evidence for the maintenance of folded structures in solution, in these hybrid sequences. The result presented in this thesis suggests that it should be possible to construct designed synthetic peptides, which can undergo transitions between two distinct and energetically favourable conformational states. The ability to design peptide sequences that can undergo switching between helical and β-hairpin states, or between hairpin structures with variations in connecting loop length may prove valuable in providing further insights into the factors influencing conformational dynamics.

The insertion of ω- amino acid residues as guests into host α-peptide sequences permits expansion of the range of polypeptide secondary structures. The term ω- amino acid is used to refer to the entire family of residues generated by homologation of the backbone of α - amino acid residues. This explores the consequences of insertion of substituted β-residues (β3) , unsubstituted β-residues , unsubstituted γ-residues (gamma aminobutyric acid) and unsubstituted δ-residues (delta aminovaleric acid) into host α -peptide sequences. Chapter 1 provides an introduction to the conformational properties of β-peptides and reviews current literature on the structural features of peptides containing ω-amino acid residues. The available crystallographic information is summarized. The conformational properties of β- residues may be described by three degrees of torsional freedom : φ (N – Cβ) , θ (Cβ -Cα) and ψ (Cα-CO). Similarly, the conformational properties of γ -residues is based on four torsional parameters ( φ , θ1 , θ2, ψ) and the conformational properties of δ - residues is based on five degrees of freedom ( φ , θ1 , θ2, θ3,ψ). The rational use of β -residues in peptide design requires an understanding of the nature of local conformations, which are readily accessible. The conformational space for β -residues can be represented in a three dimensional plot. The observed distribution of φ , θ and ψ values for β -residues in peptide crystal structures presented in this section permits a correlation - between the torsion angle θ and the secondary structure context. The gauche (g+ and g ) conformations induce helical folding and the trans conformation is generally observed in the strands of a hairpin. The most striking feature of hybrid sequences is the observation of novel hydrogen bonded rings in peptide structures. Chapter 2 describes the effects of insertion of β-residues into specific positions in the strand segments of designed peptide hairpins. Insertion of β -residues into the strands of a hairpin changes the orientation of peptide bonds, resulting in a “polar sheet” arrangement. The conformational analysis of three designed peptide hairpins composed of α/β - hybrid segments are described: Boc-Leu-βPhe-Val-DPro-Gly- Leu-βPhe-Val-OMe (BBH8) , Boc-βLeu- Phe-βVal-DPro-Gly-βLeu-Phe-βVal-OMe (BAB8) and CF3COO-H3N+-Leu-Val-Val-βPhe-DPro-Gly-βPhe-Leu-Val-Val-OMe (BHFF10). All the peptides have been characterized by 500 MHz 1H-NMR spectroscopy and several crucial long range NOEs confirm a predominant population of β-hairpin conformations in CD3OH. X-ray diffraction studies on single crystals of peptide BBH8 reveal a β-hairpin conformation, stabilized by three cross-strand hydrogen bonds and a Type II′β-turn at the DPro-Gly turn segment. Designed β-hairpin peptide scaffolds may be used to probe cross-strand sidechain interactions in β-sheet structures. A previously reported peptide β-hairpin, Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe exhibited an anomalous far UV CD spectrum, which was interpreted in terms of interactions between facing aromatic chromophores, Phe 2 and Phe 7 (Zhao, C.; Polavarapu, P.L.; Das,C. and Balaram, P. J. Am. Chem. Soc., 2000, 122, 8228-8231). In BBH8 and BHFF10 the two cross-strand βPhe residues are at non-hydrogen bonding positions, with the benzyl sidechains pointing on opposite faces of the β- sheet. BBH8 yields a “hairpin –like” CD spectrum, with a minimum at 224 nm. The CD spectrum of BAB8 reveals a negative band at 234 nm and a positive band at 221 nm suggestive of an exciton split doublet. BHFF10 yields a “hairpine-like” CD spectrum, with a negative band at 220 nm. Chapter 3 describes the synthesis and conformational characterization of three hybrid decapeptides : Boc-Leu-Val-βGly-Val-DPro-Gly- Leu-βGly -Val-Val-OMe (BHB10), Boc-Leu-Val-γAbu-Val-DPro-Gly- Leu-γAbu -Val-Val-OMe (BHC10) and Boc- Leu-Val-δAva-Val-DPro-Gly- Leu-δAva -Val-Val-OMe (BHD10). These peptides were designed to systematically investigate the effect of insertion of additional methylene groups into the strands of a hairpin. The incorporation of additional carbon atoms changes the local polarity of the strands. 500 MHz NMR studies establish that BHB10 and BHD10 adopt predominantly β- hairpin conformations in methanol, with interstrand registry established by observation of long range NOEs. The observation of both DPro 4 (CαH) ↔ Gly 5 (NH) and Gly 5 (NH) ↔ Leu 6 (NH) NOEs provides evidence for a Type II ′β - turn for all the hairpins. In BHC10, no long range NOEs were observed. However, X-ray diffraction studies in single crystals reveal a β- hairpin conformation, nucleated by a DPro-Gly Type II′β-turn. Chapter 4 describes an attempt to incorporate one or two ω amino acid residues in the turn region of a potential hairpin, in order to assess the effect of expansion of the nucleating turn. The DPro-LPro segment has been shown to stabilize β-hairpin conformations in both cyclic (Shankaramma,S.C.; Moehle, K. ; James, S.; Vrijbloed, J.W.; Obrecht,D and Robinson, J.A. Chem Commun. 2003,1842-1843) and acyclic sequences ( Raj Kishore Rai ; S.Raghothama and P. Balaram , unpublished results) . In the present study the following turn segments have been considered: βDPro -αLPro , βLPro -αLPro and βLPro -αDPro. The synthesis and conformational analysis of three octapeptide sequences -Boc-Leu-Phe-Val-βDPro-αLPro-Leu-Phe-Val-OMe (βDPαLP8), Boc-Leu-Phe-Val-βLPro-αLPro-Leu-Phe-Val-OMe (βLPαLP8)and Boc-Leu-Phe-Val-βLPro-αDPro-Leu-Phe-Val-OMe (βLPαDP8) are described. In the βDPro-αLPro peptide, NMR evidence clearly supports a β-hairpin conformation, with a nucleating hybrid βα turn stabilized by a C11 (4 →1) hydrogen bond. In the other two octapeptides, no evidence for folded structures was obtained. These results suggest that nucleating turn formation is facilitated only in the heterochiral βD-αL case. Further expansion of the turn segment in potential hairpins has been investigated by inserting two contiguous β-residues into the center of a host α-peptide sequence. The conformational studies on two synthetic hexapeptides, Boc-Leu-Phe-βDPhe-βLPro-Phe-Leu-OMe (βDFβLP6) and Boc-Leu-Phe-βLPhe-βLPro-Phe-Leu-OMe (βLFβLP6) suggest that the βDPhe-βLPro segment is capable of forming a C12 turn in methanol. Two octapeptide sequences, Boc-Leu-Val-Leu-βDPhe-βLPro-Leu-Phe-Val-OMe (βDFβLP8N) and Boc-Leu-Val-Val-βDPhe-βLPro-Leu-Val-Val-OMe (βDFβLP8V) have also been investigated to probe the possible formation of hairpin structures. In these cases, spectroscopic analysis is hampered by the presence of multiple conformations, because of the tendency of the βDPhe-βLPro bond to exist in both cis and trans conformations. NMR studies on the conformational properties of a hexapeptide Boc-Leu-Val-βDPro-βLPro-Leu-Phe-OMe (βDPβLP6) in CDCl3 reveal that in the major conformer the Val 2(NH) ↔ Leu 5 (NH) NOE is observed, suggesting the presence of a 12-membered hydrogen bonded turn. A ββ - segment can give rise to two types of hydrogen bonded rings , 10 – membered (C10) and 12- membered (C12). In an attempt to generate C10 turns, an N-methylamino acid has been inserted next to a ββ - segment, preventing the formation of the 12 – membered turn. In such a situation formation of a 10-membered turn, with reverse hydrogen bond directionality, may be facilitated. The conformational properties of Boc-Leu-Val-βDPhe-βLPro-(N-Me) Leu- Phe-OMe (βDFβLPNMeL6) has been studied by 500 MHz NMR spectroscopy. The data suggests the formation of a C11 turn at the βLPro- (N-Me) Leu segment in CDCl3-DMSO mixtures, instead of formation of a C10 turn at the βDPhe -βLPro segment. Studies on the peptide Boc-Leu-Phe-Val-βLPro-(N-Me) Leu-Leu-Phe-Val-OMe (βLPNMeL8) also suggest the absence of turn formation and folded structures. In hybrid sequences, an important question to be addressed is whether ω amino acids can be accommodated into helical structures. Two contiguous β- residues have been inserted into a helical sequence. The conformational properties of a 11- residue peptide, Boc-Val-Ala-Phe-Aib-βVal-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA11) are described in Chapter 5. This sequence was based on the parent α- peptide Boc-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-Val-Ala-Phe-Aib-OMe, which adopted a complete helical conformation in crystals (Aravinda, S.; Shamala, N.; Das, C .; Sriranjini, A.; Karle, I.L. and Balaram, P. J. Am. Chem. Soc. 2003, 125, 5308-5315). 500 MHz 1H-NMR studies establish a continuous helix over the entire length of the peptide in CDCl3 solution , as evidenced by diagnostic nuclear Overhauser effects. The molecular conformation in crystals reveals a continuous helical fold, stabilized by seven intramolecular hydrogen bonds. The characterization of two synthetic octapeptides Boc-Val-Ala-βPhe-Aib-Val-Ala-βPhe-Aib-OMe (VAβFU8) (βPhe residues have been incorporated at (i /i+4 positions) and Boc-Val-Ala-βPhe-Aib-βPhe-Ala-Val-Aib-OMe (βFUβF8) (βPhe residues have been incorporated at (i /i+2 positions) is also presented. NMR data suggests the retention of helical conformation in both the peptides. In order to delineate the conformations of hybrid peptides with three contiguous β-residues, two peptides have been synthesized Boc-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA10) and Boc-Val-Ala-Phe-Aib-βGly-βLeu-βPhe-Aib-Val-Ala-Phe-Aib-OMe (ABA12). NMR studies in chloroform support continuous helical conformation in the decapeptide.

A trinucleotide repeat (TNR) is a 3 base pair (bp) DNA sequence tandemly repeated in an array. In humans, TNR sequences have been found to be associated with at least 14 severe neurological diseases including Huntington disease, myotonic dystrophy and several of the spinocerebellar ataxias. Such diseases are caused by an expansion of the repeat sequence beyond a threshold length and are characterized by non-Mendelian patterns of inheritance which lead to genetic anticipation. Although the mechanism of the genetic instability in these arrays is not yet fully understood, various models have been suggested based on the in vitro observation that TNR sequences can form secondary structures such as pseudo-hairpins. In order to investigate the mechanisms responsible for instability of TNR sequences, a study was carried out on Escherichia coli cells in which TNR arrays had been integrated into the chromosomal lacZ gene. This genetic assay was used to identify proteins and pathways involved in deletion and/or expansion instability. Deletion instability was clearly dependent on orientation of the TNR sequence relative to the origin of replication. Interestingly, it was found that expansion instability is not dependent on the orientation of the repeat array relative to the origin of replication. The replication fork reversal pathway and the RecFOR mediated gap repair pathway were found to have no statistically significant influence on the instability of TNR arrays. However, the protein UvrD was found to affect the deletion instability of TNR sequences. The roles of key helicase genes were investigated for their effects on instability of chromosomal CTG•CAG repeats. Mutation of the rep gene increased deletion in the CTG leading-strand orientation of the repeat array, and expansion in both orientations - destabilizing the TNR array. RecQ helicase was found to have a significant effect on TNR instability in the orientation in which CAG repeats were present on the leading-strand relative to the origin of replication. Mutation of the recQ gene severely limited the number of expansion events in this orientation, whilst having no effect on deletions. This dependence of expansions on RecQ was lost in a rep mutant strain. In a rep mutant expansions were shown to be partially dependent on the DinG helicase. All together, these results suggest a model of TNR instability in which expansions are due to events occurring at either the leading or lagging strand of an arrested replication fork, facilitated by helicase action. The identity of the helicase implicated is determined by the nature of the arrest.

Inhibiting protein-protein interactions to achieve a therapeutically desired effect has been a goal in the field of drug discovery for decades. Recently, advances in peptidomimetics have led researches to the use of cyclized peptides to achieve this goal. Cyclization of linear peptides restricts the number of conformations of the peptide, increasing the peptide's affinity to binding to the desired target. Cyclization also stabilizes the peptide, allowing the peptide to be resistant to proteases. This study explores the optimization of solution phase synthesis of an important integrin-mediated cell adhesion cyclic peptide for the therapeutic inhibition of multiple myeloma, cHYD1. cHYD1 was originally synthesized via solid phase peptide synthesis, and the need for a scaled up synthesis version was needed after positive bioactivity results were obtained. Chapter 3 includes the molecular modeling exploration of a possible new mechanism to which cyclized peptides could work, in which, rather than a recognition and non-recognition strand being implemented, a specific directional face is used for protein-protein interaction. This was done with the implementation of an antagonistic cyclic peptide to replace human growth hormone in its interaction with the human growth hormone receptor, and the subsequent di-cyclic peptide agonist.

L'ARN s'est longtemps vu attribuer un simple role de transmission entre l'ADN, garant de l'information genetique, et les proteines, assurant les fonctions et donc la survie cellulaire. Ce n'est qu'avec les decouvertes des ARNs de transfert dans les annees 70, puis des ribozymes dans les annees 80, qu'il a ete realise que l'ARN pouvait assurer ces deux roles : l'information genetique est stockee dans sa sequence lineaire, et l'adoption de structures tridimensionnelles complexes rend possible une activite catalytique. Depuis, de nouvelles fonctions de l'ARN n'ont cesse d'etre decouvertes, a tous les niveaux de regulation de l'expression genique entre autres. La majorite de ces fonctions repose sur la structuration tridimensionnelle d'ARNs simple brin.Dans ce travail, differents aspects de la structuration de l'ARN sont abordes, toujours en utilisant la technique de mesure de forces sur molecules uniques par piegeage optique. Dans un premier temps, une etude comparative d'une structure secondaire modele, le hairpin dans ses formes ARN et ADN, a ete realisee. La question de l'interaction d'une structure secondaire avec une proteine helicase (DbpA) a ensuite ete abordee. Enfin, dans le cadre plus general d'une etude sur l'assemblage du ribosome, nous avons debute le developpement d'une nouvelle methode d'analyse des structures secondaires. Cette methode repose sur le suretirement d'un hybride ARN ribosomique / ADN. / Traditionally, RNA has been considered as a mere intermediate between DNA, keeper of the genetic information, and proteins, which assume cells self-sustenance. With the discoveries of the transfert RNA in the 70s, and of the ribozymes in the 80s, RNA took on both roles: it can store information in its linear sequence, and tridimensional structuration enables catalytic functions. Since then, numerous roles devoted to RNA have been discovered, particularly for gene expression regulation. Most of these functions rely on tridimensional structuration of single stranded RNA. In this work, we used an optical tweezers setup to study several aspects of RNA structuration by single molecule force measurement. In a first part, we compared the dynamic behaviour of a model secondary structure made of either RNA or DNA, the hairpin. Then we considered the interaction of a secondary structure with a protein, the RNA helicase DbpA. Finally, within a wider study of ribosome assembly, we worked on the development of a new method to study tridimensional structuration. This method relies on the overstretching of a hybrid ribosomal RNA / DNA molecule.

Synthetic construction of protein molecules has been widely pursued over the last two decades. A primary goal behind de novo protein design has been to build minimal systems by capturing the essential features of protein structures. Such minimal models can be used to understand underlying principles governing folding, structure, and function of proteins molecules. Several approaches envisioning successful construction of synthetic proteins have been described over the years, some of them being admirably successful (DeGrado et al, 1999; Richardson et al> 1992; Baltzer, 1998). Specific patterning of polar and apolar residues in synthetic sequences has been widely used to achieve designed polypeptide structures like helix bundles (DeGrado et ah, 1999) and (3-sheets (Smith and Regan, 1997; Lacroix et a/., 1998), with reliance on hydrophobic driving forces for folding. Our laboratory has been pursuing a distinctly alternative approach, that employs stereochemically constrained amino acids to generate specific secondary structures which can then be assembled into composite structures by appropriately chosen linking segments. This approach, which involves linking prefabricated modules of secondary structures can be termed as a "Meccano set" approach to protein design (Balaram, 1992). The studies embodied in the present thesis describe attempts at construction of synthetic polypeptide motifs using the stereochemically directing influence of conformationally constrained amino acid residues, such as DPro or Aib (α-aminoisobutyric acid). This thesis is subdivided into 8 chapters, with Chapter 1 providing a perspective of the field of protein design. Subsequent chapters (2-8) describe studies directed towards the specific goal of construction of polypeptide motifs. Chapter 2 describes synthesis and conformational characterization of two octapeptides Boc-Leu-Val-Val-DPro-LAla-Leu-Val-Val-OMe (1) and Boc-Leu-Val-Val-DPro-DAla-Leu-Val-Val-OMe (2), designed to investigate the effect of specific β-turn stereochemistry on β-hairpin structures. 500 MHz NMR studies establish that both peptides 1 and 2 adopt predominantly β-hairpin conformations in chloroform and methanol solutions, with interstrand registry established by observation of long-range nuclear Overhauser effects (NOEs). Specific NOEs provide evidence for a type II' β-turn conformation for the DPro-LAla segment in 1, while the NMR data suggest that a type I' DPro-DAla β-turn conformation predominates in the peptide 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt β-hairpin conformations nucleated by a type II’ β-turn across DPro-LAla and stabilized by 3 cross strand hydrogen bonds. These designed β-hairpins with defined tight turns produce characteristic vibrational circular dichroism (VCD) patterns, demonstrating the utility of VCD as a probe for conformational analysis of β-hairpins. In Chapter 3, we present conformational analysis on designed β-hairpin sequences incorporating a 'Phe-Phe' residue pair at a non-hydrogen bonding position. Two octapeptides Boc-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe and Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe were synthesized and conformationally characterized by 500 MHz NMR spectroscopy. Specific NOEs observed in solution provide conclusive evidence favoring specific orientation effects pertaining to the 'Phe-Phe' pair. The peptides exhibited anomalous electronic CD, which has been explained in terms of aromatic contributions by the side chain chromophores. Interestingly, the VCD patterns obtained for these peptides were almost identical to those obtained for other β-hairpins, described in Chapter 2. Chapter 4 describes the synthesis and conformational analysis of designed decapeptide sequences with centrally located DPro-Xxx β-trun segments. Two sequences Boc-Met-Leu»Phe-Val'DPro-Ala-Leu-Val-Val-Phe-OMe (1) and Boc-Met-Leu-Val-Val-^ro-Gly-Leu-Val-Val-Phe-OMe (2) were designed to study the effect of chain length elongation, of β-strands, on designed β-hairpin structures. 500 MHz NMR studies establish β-hairpin folds in both these sequences, with strand segments aligned even at the termini of the structures. Multi-stranded, antiparallel β-sheet structures can be generated by successive placement of β-hairpin sequences in a single polypeptide chain. The successful construction of three stranded β-sheet structures is described in Chapter 5 of this dissertation. A 14-residue peptide Boc-Leu-Phe-Val-DPro-Gly-Leu-Val-Leu-Ala-DPro-Gly-Phe-Val-Leu-OMe (LFV14) was designed such that it is composed of three strand segments linked by two DPro-Gly turn segments. The peptide showed excellent solubility in apolar media, permitting detailed conformational analysis by 500 MHz NMR spectroscopy in organic solvents. Observation of long-range, interstrand NOEs, diagnostic of multiple hairpin structures, provides conclusive evidence for a predominantly populated three stranded β-sheet structure in solution. Extension of this strategy has been described in which an 18-residue peptide, Arg-Gly-Thr-Ile-Lys-DPro-Gly-Val-Thr-Phe-Ala-DPro-Ala-Thr-Lys-Tyr-Gly-Arg, was designed with enhanced solutility in water to probe (β-sheet structure formation in aqueous and mixed aqueous-methanol systems. NMR data provided conclusive evidence in favor of the desired structure being significantly populated in methanol and methanol-water mixtures (50 %, v/v). In water, spectroscopic evidence suggests that the long-range order expected of a three-stranded structure is lost, possibly due to water invading the interstrand hydrogen bonds. Successful construction of a four-stranded antiparallel β-sheet structure has been demonstrated in Chapter 6. A 26-residue peptide Arg-Gly-Thr-Ile-Lys»DPro-Gly-Ile-Thr- Phe-Ala-DPro-Ala-Thr-Val-Leu-Phe-Ala-Val-DPro-Gly-Lys-Thr-Leu-Tyr-Arg was designed to have four strand segments linked by three DPro-Xxx turn segments. The peptide exhibited excellent NMR properties permitting structure determination by analysis of NOE data, which revealed that a four stranded β-sheet structure is indeed populated in methanol. Structural studies on this peptide in mixed methanol-water established that the four stranded β-sheet is appreciably populated at a composition of 50 % (v/v) methanol-water mixture, with the β-sheet structure still detectable even at a composition of 70 % water-30 % methanol. In a completely aqueous environment, the β-sheet structures is significantly disrupted, presumably due to solvent invasion. The nucleating β-turns, however, appear to have retained their structural integrity even in this competitive environment. Chapter 7 describes the insertion of L-Lactic acid (Lac), a hydroxy acid, into polypeptide helices stabilized by a-aminoisobutyricacid (Aib). This study was undertaken to investigate the effect of hydrogen bond deletion on peptide helices. Crystal structure determination of three oligopeptides containing Lac residues has been performed. Peptide 1, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Ala-Leu-OMe, and peptide 2, Boc-Val-Ala-Leu-Aib-Val-Lac-Leu-Aib-Val-Leu-OMe adopt completely helical conformations in the crystalline state, with the Lac(6) residue comfortably accommodated in the center of a helix. NMR studies of peptide 1 and its all amide analog 4, Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe, provide firm evidence for a continuous helical segment in both the cases. In a 14-residue peptide 3, Boc-Val-Ala-Leu-Aib- Val- Ala-Leu- Val- Ala-Leu- Aib-Val-Lac-Leu-OMe, residues Val( 1 )-Leu( 10) adopt a helical conformation, which is terminated by formation of a Schellman motif, with Aib(ll) as the site of chiral reversal. The loss of the hydrogen bond at the C-terminus appears to facilitate the chiral reversal at Aib(l 1). In the final section of this thesis, Chapter 8, successful construction of a synthetic motif containing two distinct elements of secondary structure, a (β-hairpin and a helix, has been described. The design of a 17-residue peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-DPro-Gly-Leu-Phe-Val-OMe, BH17, is based on a modular approach, in which previously characterized β-hairpin (Leu-Phe-Val-DPro-Gly-Leu-Phe-Val) and helix (Val-Ala-Leu-Aib-Val-Ala-Leu) modules are linked by a Gly-Gly linker. The positioning of the achiral Gly residue at position 8 facilitates termination of the potential helical segment (residues 1-7) by formation of a Schellman motif. Gly(9) is anticipated to be the sole conformationally flexible residue. NMR studies on BH17 indicated the presence of both the helix (residues 1-7) and the β-hairpin (residues 10-17) structures in the sequence, with four major conformational possibilities at the linking segment. Crystal structure determination of BH17 revealed that the two elements of structure are approximately arranged in an orthogonal fashion. The crystal structure validates the original premise that a modular assembly strategy may be viable for the construction of larger synthetic structures. Chapter 9 summarises the major results of this thesis. (For formulae, please refer "pdf" format)

No description available.

The study of synthetic peptides aid in improving our current understanding of the fundamental principles for the de novo design of functional proteins. The investigation of designed peptides has been instrumental in providing answers to many questions ranging from the conformational preferences of amino acids to the compact folded structures and also in developing tools for understanding the growth and formation of the protein secondary structures (helices, sheets and turns). In addition, the self-assembly of peptides through non-covalent interactions is also an emerging area of growing interest. The design of peptides which can mimic the protein secondary structures relies on the use of stereochemically constrained amino acid residues at select positions in the linear peptide sequences, leading to the construction of protein secondary structural modules like helices, hairpins and turns. The use of non-coded amino acid residues with strict preferences for adopting particular conformations in the conformational space becomes the most crucial step in peptide design strategies. In addition the crystallographic characterization and analysis of the sequences provides the necessary optimization of the design strategies. The crystallographic characterization of designed peptides provides a definitive and conclusive proof of the success of a design strategy. Furthermore, the X-ray structures provide an atomic view of the interactions, both strong and weak, which govern the growth of the crystal. The information on the geometric parameters and stereochemical properties of a series of peptides, through a systematic study, provides the necessary basis for further scientific investigation, like the molecular dynamics and can also aid in improving the force field parameters meant for carrying out molecular simulations. This can be further complemented by constructing biologically active peptide sequences. The focus of this thesis is to characterize crystallographically the conformational and structural aspects of peptide nanotubes and encapsulated water wires and the β-hairpin peptide models of β-sheets. The systematic study of a series of pentapeptide and octapeptide sequences, containing Aib and D-amino acid residues incorporated at strategic positions, establish the conformation and structural properties of designed peptides as mimics of protein secondary structures and hydrophobic tubular peptide channels and close-packed forms. The structures reported in this thesis are given below: 1 Boc-DPro-Aib-Leu-Aib-Val-OMe (DPUL5) C30H53N5O8 2 Boc-DPro-Aib-Val-Aib-Val-OMe (DPUV5a) C29H51N5O8 .(0.5) H2O 3 Boc-DPro-Aib-Val-Aib-Val-OMe (DPUV5b) C27H51N5O8 .(0.17) H2O 4 Boc-DPro-Aib-Ala-Aib-Val-OMe (DPUA5) C27H47N5O8 5 Boc-DPro-Aib-Phe-Aib-Val-OMe (DPUF5) C33H48N5O8 6 Boc-Pro-Aib-DLeu-Aib-DVal-OMe (PUDL5) C30H53N5O8 7 Boc-Pro-Aib-DVal-Aib-DVal-OMe (PUDV5a) C27H51N5O8 .(0.17) H2O 8 Boc-Pro-Aib-DVal-Aib-DVal-OMe (PUDV5b) C27H51N5O8 . 2H2O 9 Boc-Pro-Aib-DAla-Aib-DVal-OMe (PUDA5) C27H47N5O8 10 Boc-Pro-Aib-DPhe-Aib-DVal-OMe (PUDF5) C33H48N5O8 11 Ac-Phe-Pro-Trp-OMe (FPW) C28H32N4O5.(0.33)H2O 12 Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (DPLP8) C56H84N8O1 1 .(0.5) H2O 13 Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (YDPP8) C56H83N8O12 .(1.5) H2O 14 Boc-Leu-Val-Val-DPro-ψPro-Leu-Val-Val-OMe (PSIP8) C56H84N8O11S1 .(1.5) H2O 15 Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (DPPV8) C48H84N8O11 16 Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (DPUF8) C57H88N8O11.(1.5) H2O 17 Piv-Pro-ψH,CH3Pro-NHMe (PSPL3) C22H37N3O5S1 18 Boc-Leu-Val-Val-Aib-DPro-Leu-Val-Val-OMe (UDPV8) C47H84N8O11.2(C3H7NO) 19 Boc-Leu-Phe-Val-DPro-Ala-Leu-Phe-Val-OMe (BH1P8) C54H78N8O11.H2O 20 Boc-Leu-Phe-Val-DPro-Aib-Leu-Phe-Val-OMe (DPUFP8) C55H84N8O11. (0.5) H2O 21 Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (YDPPP8) C56H83N8O12. (1.5) H2O The crystal structure determination of the peptides presented in this thesis provides a wealth of information on the folding patterns of the sequences, in addition to the characterization of many structural and geometric properties. In particular, the study sheds light on the growth and formation of peptide nanotubes and the structure of encapsulated water wires, and also the structural details of Type I′ and Type II′β-turn nucleated hairpins. The study provides the backbone and side chain conformational parameters of the sequences, highlighting the varied conformational excursions possible in the peptide molecules. The thesis is divided into 6 chapters and one appendix. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α-amino acid residues and the major secondary structures of α-peptides, namely α-helix, β-sheet and β-turns. The basic structural features of helices and sheets are given. A brief introduction to polymorphism and weak interactions is also presented, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 is divided into two parts. PART 1 describes the crystal structures of a series of eight related enantiomeric peptide sequences (Raghavender et al., 2009; Raghavender et al., 2010). The crystal structures of four sequences with the general formula Boc-DPro-Aib-Xxx-Aib-Val-OMe (Xxx = Ala/Val/Leu/Phe) and the enantiomeric sequences provided a set of crystal structures withdifferent packing arrangements. The structure of the peptide with Xxx = Leu revealed a nanotube formation with the Leu lining the inner walls of channel. The channels were found to be empty. The sequence with Xxx = Val revealed a solvent-filled water channel.Investigation of the water wire structures on the diffraction data collected on the same crystal over a period of time revealed the existence of two different kinds of water wires in thechannels. Comparison with the peptide tubular structures available in the literature and the water structure inside the aquaporin channels are contrasted. Close-packed structures are observed in the case of Xxx=Ala and Phe. The backbone conformations are essentially identical. Enantiomeric sequences also revealed similar structures. Polymorphic forms were observed in the case of DVal(3) containing sequence. One form is observed to have water-filled channels forming a nanotube, as opposed to the close-packed structure in the polymorphic form. Crystal parameters DPUL5: C30H53N5O8; P65; a = b = 24.3673 (9) Å, c = 10.6844 (13) Å; α = β = 90°, γ = 120°; Z = 6; R = 0.0671, wR2 = 0.1446. DPUV5a: C29H51N5O8 .(0.5) H2O; P65; a = b = 24.2920 (13) Å, c = 10.4838 (11) Å; α = β = 90°, γ = 120°; Z = 6; R = 0.0554, wR2 = 0.1546. DPUV5b: C29H51N5O8 .(0.17) H2O; P65; a = b = 24.3161 (3) Å, c = 10.1805 (1) Å; α = β = 90°, γ = 120°; Z = 6; R = 0.0617, wR2 = 0.1844. DPUA5: C27H47N5O8; P212121; a = 12.2403 (8), b = 15.7531 (11) Å, c = 16.6894 (11) Å; Z =4; R = 0.0439, wR2 = 0.1249. DPUF5: C33H48N5O8; P212121; a = 10.3268 (8), b = 18.7549 (15) Å, c = 18.9682 (16) Å; Z = 4; R = 0.0472, wR2 = 0.1325. PUDL5: C30H53N5O8; P61; a = b = 24.4102 (8) Å, c = 10.6627 (7) Å; α = β = 90°, γ = 120°; Z = 6; R = 0.0543, wR2 = 0.1495. PUDV5a: C29H51N5O8 .(0.17)H2O; P61; a = b = 24.3645 (14) Å, c = 10.4875 (14) Å; α = β = 90°, γ = 120°; Z = 6; R = 0.0745, wR2 = 0.1810. PUDV5b: C29H51N5O8. 2H2O; C2; a = 20.7278 (35), b = 9.1079 (15) Å, c = 19.5728 (33) Å; α = γ = 90°, β = 94.207°; Z = 6; R = 0.0659, wR2 = 0.1755. PUDA5: C27H47N5O8; P212121; a = 12.2528 (12), b = 15.7498 (16) Å, c = 16.6866 (16) Å; Z = 4; R = 0.0473, wR2 = 0.1278. PUDF5: C33H48N5O8; P212121; a = 10.3354 (8), b = 18.7733 (10) Å, c = 18.9820 (10) Å; Z = 4; R = 0.0510, wR2 = 0.1526. PART 2 describes the crystallographic characterization of the tubular structure in a tripeptide Ac-Phe-Pro-Trp-OMe (FPW) sequence. The arrangement of the single-file water moleculesin the peptide nanotubes of FPW could be established by X-ray diffraction. In addition, the energetically favoured arrangement of the water wire inside the peptide channels could be modeled by understanding the construction of the peptide nanotube. In particular, the helicalmacrodipole of the peptide nanotube and the water wire dipoles prefer an antiparallel arrangement inside the peptide channels as opposed to parallel arrangements, is established by the classical dipole-dipole interaction energy calculation. In addition, the growth of thenanotubes and the arrangement of the water wires inside the channels could be correlated to the macroscopic dimensions of the crystal by the indexing of the crystal faces and contrasted with the structure of DPUV5. Crystal parameters FPW: C28H32N4O5.(0.33)H2O; P65; a = b = 21.5674 (3) Å, c = 10.1035 (2) Å; α = β = 90°, γ = 120 °; Z = 6; R = 0.0786, wR2 = 0.1771 Chapter 3 provides the crystal structures of five octapeptide β-hairpin forming sequences and a tripeptide containing a modified amino acid, with modification in the side chain (pseudo-proline, ψH,CH3Pro). The parent peptide, Boc-Leu-Phe-Val-DPro-Pro-Leu-Phe-Val-OMe (DPLP8), was observed to form a strong Type II′β-turn at the DPro-Pro segment, and the strand segments adopting a β-sheet conformation. Two molecules were observed in the asymmetric unit, inclined to each other at approximately 70°. Modification in the strand sequence Phe(2) to Tyr(2) also resulted in a hairpin with identical conformation and similar packing arrangement. The difference was in the solvent content. In both the cases the molecules were packed orthogonal with respect to each other, resulting in the formation of ribbon-like structures in three dimensions. The replacement of Phe(2) and Phe(7) with Valine residues, with the retention of DPro-Pro β-turn segment, results in an entiely different packing arrangement (parallel). Modification of Pro(5) residue of the turn segment to Aib(5) and ψPro, also results in the molecules packing orthogonally to each other. The tripeptide with a modified form of ψPro, namely ψH,CH3Pro, resulted in a folded structure with a Type VIa β-turn, with the amide bond between the Pro-ψH,CH3Pro segment adopting a cis configuration (Kantharaju et al., 2009). Crystal parameters DPLP8: C56H84N8O11 .(0.5) H2O; P21; a = 14.4028 (8), b = 18.9623 (11) Å, c = 25.4903 (17) Å, β = 105.674 ° (4); Z = 4; R = 0.0959, wR2 = 0.2251. YDPP8: C56H84N8O12 .(1.5) H2O; P212121; a = 14.4028 (8), b = 18.9623 (11) Å, c = 25.4903 (17) Å, Z = 8; R = 0.0989, wR2 = 0.2064. PSIP8: C57H86N8O11S1.(1.5) H2O; C2; a = 34.6080 (2), b = 15.3179 (10) Å, c = 25.6025 (15) Å, β = 103.593 ° (3); Z = 4; R = 0.0931, wR2 = 0.2259. DPPV8: C48H84N8O11; P1; a = 9.922 (3), b = 11.229 (4) Å, c = 26.423 (9) Å, α = 87.146 (6), β = 89.440° (6), γ = 73.282 (7); Z = 2; R = 0.1058, wR2 = 0.2354. DPUF8: C57H88N8O11 .(1.5) H2O; P21; a = 18.410 (2), b = 23.220 (3) Å, c = 19.240 (3) Å, β = 118.036 ° (4); Z = 4; R = 0.1012, wR2 = 0.2061. PSPL3: C22H37N3O5S1; P31; a = b = 14.6323 (22), c = 10.4359 (22) Å, α = β = 90°, γ = 120°; Z = 3; R = 0.0597, wR2 = 0.1590. Chapter 4 describes the crystal structure and molecular conformation of Type I′β-turn nucleated hairpin. The incorporation of Aib-DPro segment in the middle of Leu-Val-Val strands in the peptide sequence Boc-Leu-Val-Val-Aib-DPro-Leu-Val-Val-OMe results in an obligatory Type I′ turn containing hairpin. The molecular conformation and the packing arrangement of the molecules in the crystal are contrasted with the only Type I′β-hairpin reported in the literature and with a sequence where the turn residues are flipped and strand residues replaced with Phe(2) and Phe(7). Crystal parameters UDPV8: C47H84N8O11.2(C3H7NO); P21; a = 11.0623 (53), b = 18.7635 (89) Å, c = 16.6426 (80) Å, β = 102.369 (8); Z = 2; R = 0.0947, wR2 = 0.1730. Chapter 5 provides the crystal structures of three polymorphic forms of β-hairpins. The structure of BH1P8 provides new insights into the packing of hairpins inclined orthogonally to each other. The two polymorphic forms differ not only in their modes of packing in crystals but also in the strong and weak interactions stabilizing the packing arrangements. The polymorphic forms of DPUFP8 differ only in the content of the solvent in the asymmetric unit and the role it plays in bridging the symmetry related pairs of molecules. The polymorphic form YDPPP8 crystallized in a completely different space group, revealing a completely different mode of packing and also the cocrystallized solvent participating in a different set of interactions. Crystal parameters BH1P8: C54H78N8O11.H2O; P212121; a = 18.7511 (9), b = 23.3396 (11) Å, c = 28.1926 (13)Å; Z = 8; R = 0.1208, wR2 = 0.2898. DPUFP8: C55H84N8O11. (0.5) H2O; P21; a = 18.0950 (4), b = 23.0316 (5) Å, c = 18.6368 (5) Å, β = 117.471 (2); Z = 4; R = 0.0915, wR2 = 0.2096. YDPPP8: C56H83N8O12. (1.5) H2O; P21; a = 14.3184 (8), b = 18.9924 (9) Å, c = 25.1569 (14) Å, β = 105.590 (4); Z = 4; R = 0.1249, wR2 = 0.2929. Chapter 6 provides a comprehensive overview of the β-hairpin peptide crystal structures published in the literature as well as those included in the thesis. The hairpins are classified based on the residues composing the β-strands and the mode of their packing in the crystals. In the crystal structures the hairpins are observed to adopt either a Type II′ or Type I′β-turns. The indexing of the crystal faces of a few representative hairpin peptides crystallographically characterized in this thesis, provides a rational explanation for the preferential growth of the crystals in certain directions, when correlated with the strong directional forces (hydrogen bonding) and weak interactions (van der Waals, aromatic-aromatic) observed in the crystal packing. The insights gained by these studies would be highly valuable in understanding the nucleation and growth of β-hairpin peptides and the formation of β-sheet structures. Appendix I describes the Cambridge Structural Database (CSD) analysis of the conformational preferences of the proline residues found in the peptide crystal structures. The frequency distributions of the backbone φ, ψ and ω and side chain χ1, χ2, χ3, χ4 and θ torsion angles of the proline residues are calculated, tabulated and represented as graphical plots. The correlation between the backbone and endocyclic torsion angles provides for a clear evidence of the role of a particular torsion variable χ2 in deciding the state of puckering. In addition, the endocyclic bond angles also appear to be correlated, relatively strongly, with the χ2 torsion. This provides a geometrical explanation of the factors governing the puckering of the proline ring.

No description available.