Venoms, toxins and host-defence systems constitute rich sources of biologically active molecules, many of which have enormous therapeutic and biotechnological potential. In particular, peptides are often a significant component of these chemical arsenals, and are fundamentally important as biological effector molecules. The research presented in this thesis is centred on the isolation and investigation of peptides from both frogs and spiders, and endeavours to probe the important structural and mechanistic features of these bioactive compounds. The skin peptide profiles of interspecific hybrids between the green tree frog Litoria caerulea and the magnificent tree frog Litoria splendida have been investigated in a ninemonth survey. Fourteen peptides were characterised primarily using mass spectrometry, of which three had not been identified previously in the skin secretions of either parent. A number of these peptides are antibacterial agents, while others effectively inhibit the formation of nitric oxide by neuronal nitric oxide synthase. Implications for the genetics and expression of amphibian dermal peptides are also discussed. The majority of frogs of the genus Litoria contain at least one peptide in their glandular secretion capable of inhibiting the formation of nitric oxide by the enzyme neuronal nitric oxide synthase. This was proposed to occur by preventing the association of the regulatory cofactor, Ca²⁺ -calmodulin, with its binding site on the enzyme. Non-covalent binding of the amphibian peptides to calmodulin in the presence of Ca²⁺ has been confirmed using electrospray ionisation mass spectrometry, by the observation of complexes in the gas phase with a 1 : 1 : 4 calmodulin / peptide / Ca²⁺ stoichiometry. In addition, the structure and binding interactions of caerin 1.8, a potent nitric oxide synthase inhibitor, have been further probed using mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Recently a number of small, disulfide - containing neuropeptides of the signiferin and riparin families have been characterised from the skin secretion of frogs of the Crinia genus. Of these, signiferin 1 and riparin 1.1 are both ten residue peptides with similar primary sequences, however appear to have a significantly different spectrum of bioactivity. Although both act at cholecystokinin-2 receptors, signiferin 1 is smooth muscle active while riparin 1.1 is not, and instead causes proliferation of lymphocytes. The three-dimensional structures of these peptides were determined using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Both signiferin 1 and riparin 1.1 adopt β - turn type conformations, however differences in these structures may be responsible for the variation in biological activity noted for these peptides. The dermal secretions of most Australian frogs contain at least one broad-spectrum peptide antibiotic, and often a series of peptides with differing activity to afford greater protection against microbial pathogens. Solid state nuclear magnetic resonance spectroscopy studies were carried out to investigate the interaction of a number of these antibacterial peptides with anionic model membranes, and the results are compared with work previously reported using neutral lipids. It appears the peptides may have a different mode of interaction with the membranes depending upon the charge of the lipid head group. The cupiennin 1 peptides have been identified in the venom of the neotropical wandering spider, Cupiennius salei, and demonstrate potent wide-spectrum antibacterial activity. Primary sequence analysis of these peptides suggests a unique amphipathic structure distinctly different from that of other potentially helical cationic antimicrobial peptides isolated thus far. Using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations, cupiennin 1a was found to adopt an α- helical structure with a flexible central hinge region in membrane mimicking solvents. Following this, nuclear magnetic resonance spectroscopy methods were used to further probe the antibacterial and the newly identified neuronal nitric oxide synthase inhibitory activity of this peptide. / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 2006

Graduation date: 1992

Peptide YY (PYY) is a gut-derived hormone that is renowned for its effects on satiety. Reduced satiety in obese people has been attributed to low fasting and postprandial PYY levels. However, it has not been determined whether low PYY levels are the cause or the outcome of obesity. Moreover, the long-term role of PYY in regulating energy balance is unclear. Results presented in this thesis, using PYY-deficient mice (PYY-/-) and PYY transgenic mice (PYYtg) highlight that PYY indeed has an important role in regulating energy balance and glucose homeostasis in vivo. PYY knockout mice became obese with ageing or high-fat feeding linked to a hyperinsulinemic phenotype associated with hypersecretion of insulin from isolated pancreatic islets. These findings suggested that PYY deficiency may be a predisposing factor for the development of obesity and type 2 diabetes. On the other hand, PYYtg mice exhibited decreased adiposity and increased metabolism under high-fat feeding. Furthermore, PYYtg/ob mice had improved glucose tolerance and decreased adiposity. These latter studies suggested that high circulating PYY levels may protect against the development of obesity and type 2 diabetes. Interestingly, both animal models support PYY as an important regulator of the somatotropic axis. These preliminary findings prompted investigations in understanding whether low PYY levels may be a predisposing factor for the development of obesity and type 2 diabetes in human subjects. In a population of healthy human subjects that had a predisposition to the development of type 2 diabetes and obesity, fasting PYY levels were lower than in normal subjects. Moreover, low fasting PYY levels strongly correlated with decreased insulin sensitivity and high levels of fasting insulin. Collectively, these findings suggest that low circulating levels of PYY could contribute to increased adiposity, insulin resistance and type 2 diabetes. Therefore determination of PYY levels may be a method of detecting whether people are predisposed to becoming obese and insulin resistant. This work also suggests that treatments that enhance circulating PYY levels may be protective in the development of obesity and type 2 diabetes.

Cell penetrating peptides (CPPs) are short sequences of amino acids that excel in crossing the cellular membrane without inducing cytotoxicity Interest in these peptides stem from their ability to be attached, and grant their penetrating properties to, a variety of cargo In this work we have combined the application of Confocal Raman Microscopy (CRM) and Atomic Force Microscopy for the first time to examine the interactions of unlabeled Transportan (TP), one of the most well studied CPPs, with mammalian cells CRM’s capability to discriminate control and treated cell groups was verified by principal component analysis (PCA) and linear discriminant analysis (LDA) and was 93-100% accurate We’ve determined that at a concentration of 20 μM TP enters cells through a non-endocytotic mechanism, has a high affinity for the cytoplasm and membranes, and results in a significant increase in cellular stiffness Our work provides the first direct evidence of this cell-stiffening phenomenon SFTI-1, the smallest member of a bicyclic, cysteine rich class of CPPs, was examined by CRM to determine the potential role of cyclic structure on cellular uptake The peptide, along with monocyclic and linear analogs was heavy isotope labeled and incubated with mammalian cells at numerous concentrations and timespans Our work is the first SFTI-1 uptake study forgoing the use of fluorophore conjugates, which have been linked to artificial cellular uptake We demonstrate herein the absence of any CRM detectable uptake, providing the first evidence that SFTI-1 may not be a CPP Finally, CRM was applied to the discrimination of normal and basal cell carcinoma cells obtained from the same donor The use of patient matched cells avoids the normal biochemical variations that exist among individuals, ensuring that discrimination is based solely on the cell’s diseased state CRM spectra, analyzed by PCA and LDA, were capable of spectral discrimination with 100% accuracy Major differences in the cancerous cells were an increase in lipids and nucleic acids, and an overall decrease in protein We also demonstrate an enhancement in Raman signal through the use of an aluminum foil substrate, providing a practical approach for measuring cells with thin morphologies / Includes bibliography / Dissertation (PhD)--Florida Atlantic University, 2016 / FAU Electronic Theses and Dissertations Collection

Spider silk is one of the most versatile material.s in nature with great mechanical properties, exceeding some of the best man made materials. Native and synthetically produced silk has been used in a wide array of applications throughout the history of mankind including nets, bandages and cloths. It is recognized that spider silk can be a suitable replacement material for many existing materials such as ropes, body armor, parachutes and biodegradable bottles - all of which could show cost and environmental 4 benefits relative to other currently used man made materials. An added advantage to these types of applications is the potential for the products to have intrinsic antimicrobial activity. Studies have demonstrated a level of antimicrobial activity in native silk, a property that may have evolved in order to resist microbial decomposition, to protect developing eggs, and to resist decomposition or destruction by predators, parasites, or fluctuations in the environment. In this study, the novel aqueous glue coating peptides found on the silk fiber of the black widow spider, spider coating peptide 1 and 2, were investigated. Using circular dichroism, it was determined that SCP-1 and SCP-2 display predominantly alpha-helical secondary structures. In temperature gradient studies, SCP-1 is structurally stable at high temperatures while SCP-2 unfolded and lost its alpha-helical structure. The two peptides remained structurally stable both in an acidic and basic environment. This study was the first to characterize the secondary structure of the peptides found coating various silk fibers in Latrodectus hesperus, the black widow spider. The function of the SCPs is unknown but has-been hypothesized to potentially have antimicrobial properties. We investigated this role and found no significant antibacterial activity of the peptides against Escherichia coli and Bacillus subtitlis in growth studies. This study is the first to investigate the functional role of SCPs.

Indiana University-Purdue University Indianapolis (IUPUI) / Histones are highly basic proteins which when digested by trypsin are hard to analyze using mass spectrometry. Because histones are basic nuclear proteins, a nuclei prep followed by acid extraction is the best purification strategy to increase overall abundance of purified histones. Blocking the lysine residues and cleaving with trypsin is a useful technique to increase detection of histone peptides using MudPIT. In particular, carbamylation and propionylation are the best two methods to block lysine residues. Using both propionylation and carbamylation along with no treatment has been shown to increase the identification of unmodified and modified histone peptides when coupled with MudPIT analysis.

Understanding the relation between amino acid sequences and protein structures is one of the most important problems in modern molecular biology. However, due to the complexities in the protein structure, this task is really daunting. Hence, understanding the structural features of proteins and the rules of folding is central to the design of novel and more effective biomaterials. With the inception of the de novo design of synthetic mimetics for protein structural elements, the study of designed peptides is a subject of intense current research. The de novo design of polypeptide structures provides insights into the factors that govern the folding of peptides and proteins. The rational design of synthetic peptide models for secondary structural motifs in proteins depends on the ability to control the polypeptide chain stereochemistry. An approach, which seems to be useful, is the introduction of constrained genetically coded amino acids like Proline or the introduction of non-protein constrained amino acids like Aib which are capable of restricting the range of available backbone conformations of the polypeptide chain. The use of such residues would then permit the design of well defined and intended structural motifs like the β-turns which serve as chain reversal areas of the polypeptide chain. Templates incorporating multiple repeats of such conformationally constrained residues would in turn further enhance the choice of conformational parameters for the polypeptide chain towards folding. Crystal structure determination of the oligopeptides by X-ray diffraction gives insight into the specific conformational states, modes of aggregation, hydrogen bond interactions and solvation of peptides. Precise structural analysis and good characterization of geometrical parameters and stereochemical details of these molecules provide valuable inputs for peptide design and are indispensable for exploring strategies to design peptide sequences which serve as synthetic mimics for folding motifs in proteins. Many of the above points have been investigated in this thesis which incorporates study of designed peptides containing heterochiral and homochiral diproline segments followed by protein database analysis. This thesis reports results of x-ray crystallographic studies of twenty two (22) oligopeptides containing heterochiral or homochiral diproline segments. Apart from the crystal data, protein database analysis has also been carried out to investigate what actually is found in nature. Given in brackets are the compound names used in the thesis for the peptides solved. 1) Piv-DPro-LPro-NHMe ( DPPN ) [C16H27N3O3 ] 2) Piv-DPro-LPro-LVal-OMe ( DPPV ) [C21H35N3O5 . 0.09 H2O] 3) Piv-DPro-LPro-LPhe-OMe ( DPPF ) [C25H35N3O5 . H2O] 4) Piv-DPro-LPro-DAla-OMe ( DPPDA ) [C19H31N3O5] 5) Piv-LPro-DPro-LAla-OMe ( PDPA ) [C19H31N3O5] 6) Piv-DPro-LPro-LVal-NHMe ( DPPVN ) [C21H36N4O4 . H2O] 7) Piv-DPro-LPro-LLeu-NHMe ( DPPLN ) [C22H38N4O4 . 0.34H2O] 8) Piv-DPro-LPro-LPhe-NHMe ( DPPFN ) [C25H36N4O4 . H2O] 9) Piv-DPro-LPro-Aib-NHMe ( DPPUN ) [C20H34N4O4] 10) Piv-DPro-LPro-DAla-NHMe ( DPPDAN ) [C19H32N4O4] 11) Piv-DPro-LPro-DVal-NHMe ( DPPDVN ) [C21H36N4O4 .1.43 H2O] 12) Piv-DPro-LPro-DLeu-NHMe ( DPPDLN ) [C22H38N4O4 . H2O] 13) Piv-LPro-DPro-LAla-NHMe ( PDPAN ) [C19H32N4O4] 14) Piv-LPro-DPro-LVal-NHMe ( PDPVN ) [C21H36N4O4] 15) Piv-LPro-DPro-LLeu-NHMe ( PDPLN ) [C22H38N4O4 . H2O] 16) Piv-LPro-DPro-LVal-OMe ( PDPVO ) [C21H35N3O5 . H2O] 17) Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe ( PPVVN ) [C21H36N4O4 . 0.74H2O] 18) Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe ( PPLLN ) [C22H38N4O4 . H2O] 19) Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe ( PPFFN ) [C25H36N4O4 . 2 H2O] 20) Piv-LPro-LPro-LPhe-OMe ( PPFO ) [C25H35N3O5 . 0.5 H2O] 21) Piv-LPro-LPro-LVal-NHMe ( PPVN ) [C21H36N4O4 . H2O] 22) Piv-LPro-LPro-Aib-NHMe ( PPUN ) [C20H34N4O4. H2O] Results from the X-ray crystallographic analysis of the above peptides provided substantial information regarding role of diproline templates on the folding of the polypeptide chain. The thesis is divided into the following eight chapters : Chapter 1 gives a general introduction to the stereochemistry of polypeptide chains and the secondary structure classification: helices, β-sheets and β-turns. This section also provides a brief overview of the use of non standard and D-amino acids into peptide design. Discussions on DProline, puckering states of the Proline ring, diproline segments and racemic mixtures of peptides are also presented. A brief discussion on X-ray diffraction and solution to the phase problem is also given. Chapter 2 describes the structural characterization in crystals of the five following designed peptides: Piv-DPro-LPro-NHMe (DPPN), Piv-DPro-LPro-Xxx-OMe [Xxx = LVal (DPPV); LPhe (DPPF); DAla (DPPDA)] and Piv-LPro-DPro-LAla-OMe (PDPA) containing the heterochiral diproline segment with an aim towards understanding the directive influence of short range interaction on polypeptide folding. Except PDPA, in all the structures, a type II’ β-turn was observed at the DPro-LPro segment with the formation of a 4→1 intramolecular hydrogen bond between the atoms of the polypeptide backbone. In PDPA, the expected type II β-turn occurred at the LPro-DPro segment. Thus, the DPro-LPro segment preferably adopts a type II’ β-turn conformation when present at the C-terminus which is mimicked by the methyl ester group. The use of pivalyol group at the N-terminus is to ensure the trans geometry of the peptide bond between pivalyol and the first Proline. Crystal parameters DPPN: C16H27N3O3; P21; a = 10.785(1) Å, b = 15.037(1) Å, c = 11.335(1) Å; β = 109.96(1)°; Z = 4; R = 0.0388, wR2 = 0.1047. DPPV: C21H35N3O5 . 0.09 H2O; P212121; a =10.676(1) Å, b = 16.608(1) Å, c = 39.887(1) Å, Z = 12; R = 0.0688, wR2 = 0.1701. DPPF: C25H35N3O5 . H2O; P21; a = 9.538(1) Å, b = 10.367(1) Å, c = 13.102(1) Å; β = 93.04(1) °; Z = 2; R = 0.0504, wR2 = 0.1455. DPPDA: C19H31N3O5; P21; a = 11.269(1) Å, b = 9.945(1) Å, c = 18.550(2) Å; β = 97.46(1)°; Z = 4; R = 0.0563, wR2 = 0.1249. PDPA: C19H31N3O5; P212121; a = 9.043(1) Å, b = 10.183(2) Å, c = 23.371(1) Å; Z = 4; R = 0.0753, wR2 = 0.1603. Chapter 3 describes the crystal structures of the four following designed peptides containing the heterochiral diproline segment followed by a L-residue or an achiral amino acid residue like Aib : Piv-DPro-LPro-Xxx-NHMe [Xxx = LVal (DPPVN); LLeu (DPPLN); LPhe (DPPFN) and Aib (DPPUN)]. In the first three peptides the DPro-LPro segennt adopts a type II’ β-turn conformation with the formation of a type I β-turn at the LPro-Xxx segment. The peptide backbone overall therefore adopts a consecutive β-turn structure. When the L-amino acids at the C-terminus are replaced by the achiral amino acid Aib, the overall folded structure adopted by the peptide backbone still remains unchanged with the formation of a consecutive β-turn. All the structures are stabilized by two intramolecular 4→1 hydrogen bonds between the C=O group and the nitrogen atom of the polypeptide backbone. Crystal parameters DPPVN: C21H36N4O4 . H2O; P21; a = 9.386(1) Å, b = 12.112(1) Å, c = 10.736(1) Å; β = 99.53(1) °; Z = 2; R = 0.0528, wR2 = 0.1337. DPPLN: C22H38N4O4 . 0.34H2O; P21; a =9.231(1) Å, b = 17.558(1) Å, c = 15.563(1) Å; β = 91.94(1) °; Z = 4; R = 0.0555, wR2 = 0.1422. DPPFN: C25H36N4O4 . H2O; P212121; a = 10.473(1) Å, b = 15.980(1) Å, c = 15.994(1) Å; Z = 4; R = 0.0620, wR2 = 0.1826. DPPUN: C20H34N4O4; P212121; a = 10.571(2) Å, b = 11.063(1) Å, c = 18.536(1) Å; Z = 4; R = 0.0578, wR2 = 0.1256. Chapter 4 describes the crystal structures of the seven designed peptides containing heterochiral diproline segment. Three of these contain sequences of the type DPro-LPro-DXxx [DXxx = DAla (DPPDAN); DVal (DPPDVN); DLeu (DPPDLN)] and three contains the enantiomeric peptides of the ones that are mentioned earlier in sequences of the type LPro-DPro-LXxx [LXxx = LAla (PDPAN); LVal (PDPVN); LLeu (PDPLN)]. In order to investigate the effect of the C-terminal protecting group, a final peptide Piv-LPro-DPro-LVal-OMe (PDPVO) was crystallographically characterized. All the peptides containing the DXxx residues adopted different backbone conformations. For DAla, a structure simultaneously having a β-turn and an α-turn was obtained which is the first example in designed peptides of an isolated α-turn. In the case of DVal, an open / extended structure devoid of any intramolecular hydrogen bonding was obtained whereas for DLeu, type II β-turn occurred at the LPro-DLeu segment instead of the expected type II’ turn at the DPro-LPro segment. In the case of enantiomeric peptides, all the three peptides adopted folded structures with exact mirror image conformation being generated for LAla and nearly identical mirror image conformation in the case of LLeu. The enantiomeric peptide of DVal which contained LVal residue following the diproline segment also adopted a folded conformation with the formation of type II β-turn at the LPro-DPro segment as expected. X-ray crystallographic characterization of PDPVO resulted in the peptide adopting an overall extended / open structure. Thus, the chirality of the C-terminal residue seems to have a profound effect on the conformation of the heterochiral diproline segments. The role of the C-terminal protecting group cannot also be undermined. Crystal parameters DPPDAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(1) Å, c = 9.961(1) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0728, wR2 = 0.1528. DPPDVN : C21H36N4O4 .1.43 H2O; P212121; a = 8.744(8) Å, b = 11.609(1) Å, c = 23.577(2) Å; Z = 4; R = 0.0625, wR2 = 0.1856. DPPDLN : C22H38N4O4 . H2O; P212121; a = 10.531(3) Å, b = 11.659(3) Å, c = 20.425(6) Å; Z = 4; R = 0.0444, wR2 = 0.1239. PDPAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(2) Å, c = 9.961(2) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0745, wR2 = 0.1572. PDPVN : C21H36N4O4; P212121; a = 9.743(1) Å, b = 11.423(1) Å, c = 21.664(3) Å; Z = 4; R = 0.0803, wR2 = 0.1899. PDPLN : C22H38N4O4 . H2O; P212121; a = 10.462(4) Å, b = 11.572(4) Å, c = 20.262(7) Å; Z = 4; R = 0.0968, wR2 = 0.2418. PDPVO : C21H35N3O5 . H2O; P212121; a = 8.784(4) Å, b = 11.587(5) Å, c = 23.328(1) Å; Z = 4; R = 0.0888, wR2 = 0.1465. Chapter 5 describes the crystal structures of the three designed peptides containing racemic mixtures [Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe (PPVVN); Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe (PPLLN); Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe (PPFFN)] having the heterochiral diproline segment in their sequences and three peptides having a homochiral diproline segment [Piv-LPro-LPro-LPhe-OMe (PPFO); Piv-LPro-LPro-LVal-NHMe (PPVN); Piv-LPro-LPro-Aib-NHMe (PPUN)]. The inability of the pure enantiomers to crystallize in the case of Phe (chapter 4) invoked the use of peptide racemates for obtaining a crystal state conformation for the said compound. In all the cases, the L-enantiomer of Xxx crystallized in the asymmetric unit. A type II β-turn was obtained in the case of PPVVN at the LPro-DPro segment and a type II’ β-turn was obtained for PPLLN at the DPro-LLeu segment. in the case of Phe, an open structure devoid of any intermolecular hydrogen bonding an very similar to DPPDVN (chapter 4) was obtained. In the case of homochiral diproline segment containing peptides, PPFO crystallized with two molecules in the asymmetric unit, both of which adopted a type VIA1 hydrogen bonded β-turn conformation with a cis peptide bond between the diproline segment. In the case of Valine (PPVN) however, a structure devoid of any intramolecular hydrogen bonding was obtained. In the final peptide PPUN, a type II β-turn conformation is observed at the LPro-Aib segment. The analysis revealed that the hydration of the peptide can cause dramatic changes in its backbone conformation. In homochiral LPro-LPro sequences, the tendency to form hydrogen bonded turns competes with the formation of semi-extended polyproline structures. The results also emphasize the subtle role of sequence effects in modulating the conformations of short, constrained peptide segments. The possibility of trapping distinct conformational segments of the diproline segments in crystals by generating racemic centro-symmetric crystals in which packing effects may be appreciably different from those observed in the crystals of individual pure enantiomeric peptides has been clearly exploited in this chapter to obtain a crystal in the case of Phe. These results suggest that the energetic differences between these states is small. Conformational choice can therefore be readily influenced by environmental and sequence effects. Crystal parameters PPVVN: C21H36N4O4 . 0.74H2O; C2/c; a = 36.667(17) Å, b = 10.092(5) Å, c = 13.846(6) Å; β = 107.27(1) °; Z = 8; R = 0.1317, wR2 = 0.3141. PPLLN: C22H38N4O4 . H2O; P21/c; a = 10.555(1) Å, b = 11.687(1) Å, c = 20.108(2) Å; β = 95.47(1) °; Z = 4; R = 0.0761, wR2 = 0.2034. PPFFN: C25H36N4O4 . 2 H2O; P21/c; a = 8.883(5) Å, b = 18.811(10) Å, c = 16.033(9) Å; β = 96.28(1) °; Z = 4; R = 0.1218, wR2 = 0.2848. PPFO : C25H35N3O5 . 0.5 H2O; P212121; a = 10.199(1) Å, b = 20.702(2) Å, c = 23.970(2) Å; Z = 8; R = 0.0716, wR2 = 0.1901. PPVN : C21H36N4O4 . H2O; P212121; a = 9.454(1) Å, b = 11.119(1) Å, c = 23.021(2) Å; Z = 4; R = 0.0551, wR2 = 0.1587. PPUN: C20H34N4O4. H2O; P21; a = 6.276(1) Å, b = 14.011(2) Å, c = 12.888(1) Å; β = 96.80(1) °; Z = 2; R = 0.0475, wR2 = 0.1322. Chapter 6 describes the pyrrolidine ring puckering states of the Proline residue present in diproline segments in the peptides solved in this thesis, the Cambridge structural database (CSD) [only acyclic diproline containing peptides have been taken into account] and in a non-redundant dataset of proteins in the Protein Data Bank (PDB). The five membered pyrrolidine ring of Proline can be best characterized in terms of the following five endocyclic torsion angles χ1, χ2, χ3,χ4 and θ. Using various values of these endocyclic torsion angles the following puckering states were identified : [1] Cγ-exo (A) [2] Cγ-endo (B) [3] Cβ-exo (C) [4] Cβ-endo (D) [5] Twisted Cγ-exo-Cβ-endo (E) [6] Twisted Cγ-endo-Cβ-exo (F) [7] Planar (G) [8] Cα-distorted (H) [9] Twisted Cβ-exo-Cα-endo (I) [10] Cδ-endo (K) [11] N-distorted (L) [12] Twisted Cδ-endo- Cγ-exo (N). In the case of peptides solved in this thesis for heterochiral diproline segments, the Cγ-exo / Cβ-exo (AC) combination turns out to more preferred than the other combinations. The Cγ-endo / Cγ-endo (BB) state is the second most populated state. The overall investigation of Proline rings in peptides show that the states Cγ-exo (A), Cβ-exo (C) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. In the case of proteins, the overall percentage distribution of various combinations indicates that the AA (Cγ-exo / Cγ-exo), AE (Cγ-exo / Twisted Cγ-exo-Cβ-endo) and FF (Twisted Cγ-endo-Cβ-exo / Twisted Cγ-endo-Cβ-exo) categories are the most preferred combinations. For Proline rings in proteins, the states Cγ-exo (A), Twisted Cγ-exo-Cβ-endo (E) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. Chapter 7 describes the analysis of diproline segments in a non-redundant dataset of proteins In this chapter, the possible conformational states for the diproline segment (LPro-LPro) found in proteins taken from a non-redundant dataset has been investigated an identified with an emphasis on the cis and trans states for the peptide bond between the diproline segment. The occurrence of diproline segments in type VIA1 turns (cis Pro-Pro peptide bond) and other regular secondary structures like type III β-turns and α-helices has been studied. This has been followed up by the amino acid distribution flanking the diproline segment and the conformation adopted by Xaa-Pro and Yaa-Pro segments in proteins. It is observed that for cis Pro-pro peptide bond, the conformation adopted by the first Proline lies in PII region whereas the second Proline inevitably adopts a conformation in the Bridge region, leading to the formation of the type VIA1 β-turn structure. But in the trans case, the conformation adopted by the first Proline is overwhelmingly populated in the PII (Polyproline) and right-handed α-helical region. For position i+2, the major conformation adopted by Proline is P II and α with a substantial amount of occurrences in Bridge and the C7 (γ-turn) region. The analysis also reveals that the cis-cis configuration of the peptide bond is very rare when considering the diproline segment. With a cis-trans peptide linkage, PII-PII conformation is the most stable and favoured conformation for the Pro-Pro segment in proteins. With trans peptide bond linkage between the Proline residues, α- α and PII-Bridge conformations are equally likely for the diproline segment. The population in trans-cis and cis-trans states are comparable indicating that the energy differences between these states is small. However, trans-trans is the most populated state with a percentage occurrence of 85.43%. The analysis and comparison of conformational states for the Xaa-Pro-Yaa sequence reveals that the Xaa-Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The same is valid for Pro-Yaa segment, with the cis conformer being populated to even lesser extent. The data shows that α- α, PII-α, PII-PII and extended-PII are the most populated states for Xaa-Pro and Pro-Yaa segments as compared to PII-PII and PII-α and states observed for the Pro-Pro segment. Chapter 8 describes the analysis of single and multiple β-turns in a non-redundant dataset of proteins. The analysis on β-turns in proteins has shed a new light into the propensity values for amino acid residues at various positions of β-turns which in certain cases have undergone appreciable change in values than previously observed. One of the other notable feature of the analysis is the fact that the data displays a higher occurrence of unprimed β-turns of type I and type II as compared to their primed counterparts of type I’ and type II’ as previously observed. In fact, the results show that type I β-turn is the highest occurring turn both in isolated as well as in consecutive β-turn examples. The analysis of multiple β-turns in proteins has revealed many new categories like the (I,I+1,I+3), (I,I+2,I+3) and combination of turns which can be used for the design of the loops, especially in the case of β-hairpins. Among the multiple turns, double turns occur more frequently than the other consecutive turns like triple and quadruple turns. It is also important to note that the number of examples of a hydrogen bonded turn being followed by a hydrogen bonded turn is very less with type IV turn preceding a primed turn in most of the cases. Thus, the data available from consecutive β-turn analysis and the type-dependent amino acid positional preferences and propensities derived from the present study may be useful for modeling various single and consecutive turns, especially in designing loop regions of β-hairpins.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been widely used in many biomedical applications. Ultraviolet (200-400 nm) or visible (400-800 nm) light can interact with light-sensitive compounds called photoinitiators to form radical species that trigger photopolylmerization. Since UV light has potential to cause cell damage, visible light-mediated photopolymerization has attracted much attention. The conventional method to fabricate hydrogels under visible light exposure requires usage of co-initiator triethanolamine (TEA) at high concentration (∼200 mM), which reduces cell viability. Therefore, the first objective of this thesis was to develop a new method to form poly(ethylene glycol)-diacrylate (PEGDA) hydrogel without using TEA. Specifically, thiol-containing molecules (e.g. dithiothreitol or cysteine-containing peptides) were used to replace TEA as both co-initiator and crosslinker. Co-monomer 1-vinyl-2-pyrrolidinone (NVP) was used to accelerate gelation kinetics. The gelation rate could be tuned by changing the concentration of eosinY or NVP. Variation of thiol concentration affected degradation rate of hydrogels. Many bioactive motifs have been immobilized into hydrogels to enhance cell attachment and adhesion in previous studies. In this thesis, pendant peptide RGDS was incorporated via two methods with high incorporation efficiency. The stiffness of hydrogels decreased when incorporating RGDS. The second objective of this thesis was to fabricate hydrogels using poly(ethylene glycol)-tetra-acrylate (PEG4A) macromer instead of PEGDA via the same step-and-chain-growth mixed mode mechanism. Formation of hydrogels using PEGDA in this thesis required high concentration of macromer (∼10 wt.%). Since PEG4A had two more functional acrylate groups than PEGDA, hydrogels could be fabricated using lower concentration of PEG4A (∼4 wt.%). The effects of NVP concentration and thiol content on hydrogel properties were similar to those on PEGDA hydrogels. In addition, the functionality and chemistry of thiol could also affect hydrogel properties.

This thesis deals with the analysis of natural peptide libraries using mass spectrometry. In the course of the study, both ribosomal and non-ribosomal classes of peptides have been investigated. Microheterogeneity, post-translational modifications (PTM), isobaric amino acids and disulfide crosslinks present critical challenges in routine mass spectral structure determination of natural peptides. These problems form the core of this thesis. Chapter 2 describes an approach where chemical derivatization, in unison with high resolution LC-MSn experiments, resulted in deconvolution of a microheterogenous peptide library of B. subtilis K1. Chapter 3 describes an approach for distinction between isobaric amino acids (Leu/Ile/Hyp), by the use of combined ETD-CID fragmentation, through characteristic side chain losses. Chapters 4-6 address a long standing problem in structure elucidation of peptide toxins; the determination of disulfide connectivity. Through the use of direct mass spectral CID fragmentation, a methodology has been proposed for determination of the S-S pairing schemes in polypeptides. Further, an algorithm DisConnect has been developed for a rapid and robust solution to the problem. This general approach is applicable to both peptides and proteins, irrespective of the size and the number of disulfide bonds present. The method has been successfully applied to a large number of peptide toxins from marine cone snails, conotoxins, synthetic foldamers and proteins. Chapter 7 describes an attempt to integrate next generation sequencing (NGS) data with mass spectrometric analysis of the crude venom. This approach couples rapidly generated cDNA sequences, with high-throughput LC-ESI-MS/MS analysis, which provides mass spectral fragmentation information. An algorithm has been developed that allows the construction of a putative conus peptide database from the NGS data, followed by a protocol that permits rapid annotation of tandem MS data. The approach is exemplified by an analysis of the peptide components present in the venom of Conus amadis, yielding 225 chemically unique sequences, with identification of more than 150 sites of PTMs. In summary, this thesis presents different methodologies that address the existing limitations of de novo mass spectral structure determination of natural peptides and presents new methodologies that permit for rapid and efficient analysis of complex mixtures.