Made available in DSpace on 2014-12-17T15:42:15Z (GMT). No. of bitstreams: 1 FabioPF_TESE.pdf: 3376603 bytes, checksum: 15dfaa7fe12ca918fd7e1b98c4378dd9 (MD5) Previous issue date: 2011-09-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 ?C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media / S?ntese enzim?tica de pept?deos usando proteases tem atra?do uma enorme aten??o nos ?ltimos anos. Um desafio chave na s?ntese de pept?deos ? encontrar suportes para imobiliza??o de proteases capazes de apresentar um alto desempenho em meio aquoso, produzindo oligopept?deos sol?veis em ?gua, j? que at? o presente momento, pouco tem sido descrito usando essa estrat?gia. Dessa forma, o objetivo dessa tese foi imobilizar proteases usando diferentes m?todos (imobiliza??o por liga??o covalente, aprisionamento em g?is polim?ricos de PVA e imobiliza??o em nanopart?culas magn?ticas de Glicidil) para a produ??o de oligopept?deos derivados da lisina. Tr?s proteases foram utilizadas: tripsina, α-quimotripsina e bromela?na. De acordo com as estrat?gias de imobiliza??o associadas ao tipo de protease empregada, foi provado que os sistemas tripsina-resinas mostraram os melhores desempenhos em termos de atividade hidrol?tica e s?ntese de oligopept?deos. A atividade hidrol?tica das enzimas livres e imobilizadas foi determinada por espectrofotometria com base na mudan?a de absorb?ncia em 660 nm ? temperatura de 25 ?C (Casein method). O rendimento de oligolisina e o c?lculo do grau de polimeriza??o m?dio foram monitorados por RMN H. A protease tripsina foi covalentemente imobilizada em quatro diferentes resinas (Amberzyme, Eupergit C, Eupergit CM and Grace 192). O m?ximo rendimento de prote?na imobilizada foi 92, 82, 60, e 71 mg/g de suporte para cada resina, respectivamente. A efici?ncia desses sistemas (Tripsina-resinas) foi avaliada pela hidr?lise do substrato case?na e a s?ntese de oligolisina em meio aquoso. A maioria dos sistemas foram capazes de catalisar a s?ntese de oligopept?deos, entretanto com diferentes efici?ncias, tais como: 40, 37 e 35% para os suportes Amberzyme, Eupergit C e Eupergit CM, respectivamente, em compara??o com a enzima livre. Esses sistemas produziram olig?meros em somente 1 hora com grau de polimeriza??o m?dio mais alto que a enzima livre. Dentre esses sistemas, Eupergit CTripsina mostrou ser mais eficiente que os outros sistemas em termos de atividade hidrol?tica e estabilidade t?rmica, ao passo que n?o exibiu a mesma efici?ncia como era esperado para a s?ntese de oligolisina. Tripsina-amberzyme provou ser mais eficiente para a s?ntese de oligopept?deos, al?m de exibir um excelente reuso, mantendo 90% de sua atividade hidrol?tica e sint?tica ap?s sete reusos. As intera??es hidrof?bicas da tripsina com o suporte Amberzyme s?o respons?veis por proteger a enzima contra as fortes mudan?as conformacionais no meio reacional. Al?m disso, a alta concentra??o de grupos oxiranos na superf?cie da resina promoveu liga??es covalentes multipontuais e, consequentemente, preveniu a enzima imobilizada do processo de desor??o (Leaching process). Os resultados acima mencionados sugerem que a tripsina imobilizada nesses suportes pode ser eficientemente usada para a s?ntese de oligopept?deos em meio aquoso

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.

ΔPhe an analogue of a coded amino acid phenylalanine (Phe) residue but with double bond between Cα and Cβ atoms, is one of the well studied residue among all the dehydro amino acids, as a conformation constraining amino acid. Due to the presence of double bond Cα=Cβ, and consequent conjugation of ΔPhe ring electrons with Cα=Cβ double bond, ΔPhe gains conformation restricting (constraining) characteristics compared to coded amino acid Phe. ΔPhe which assumes an achiral residue has all its atoms restricted to an approximate plane. Apart from the conformation constraining property, the designer friendly ΔPhe residue has its ability to i) engage in side chain aromatic interactions ii) act as nuclei for C-HLO/N-HLπ weak interactions involving the side chain and/ or backbone atoms, and iii) acquire ambidextrous conformation as observed in many model peptides. It is these properties, which makes ΔPhe, a residue of intense research in the field of de novo protein secondary and super secondary design. Analysis of solid state and solution state structures of containing ΔPhe residues suggests that ΔPhe, in general induces β-bend in short peptides and 310-helical conformation in longer peptides (>4).

The thesis describes utilizing mass spectrometry and computational methods to study two groups of molecular systems: small organic molecules and oligopeptides. The gas-phase acidities were measured and the structures of the molecular species were calculated. The small molecules investigated included methylparaben, ibuprofen, and triclosan, all known to have some biological activity. The gas-phase acidity measurements made for these small molecules had the solvent and collisional gas pressures adjusted in order to observe their potential influences. The results obtained provide insight into the ion chemistry of these molecules and how the energetics may change the observed behavior of the ion as well as the resulting thermochemical properties measured. The oligopeptides studied were a family of tri-peptides in which a cysteine probe was placed within an alanine backbone. The cysteine probe was either in the L- or D- configuration in order to detect any fundamental differences among the diastereotopic peptides. Compared to the L-cysteine isomers, the D-cysteine peptides appear to display a change in gas-phase behavior and their respective dissociation profiles. These changes may have an implication of altering the biochemical properties when chirality changes in biological systems.

<div>Delayed fracture healing is a major health issue involved with aging. Therefore, strategies to improve the pace of repair and prevent non-union are needed in order to improve patient outcomes and lower healthcare costs. In order to accelerate bone fracture healing noninvasively, we sought to develop a drug delivery system that could safely and effectively be used to deliver therapeutics to the site of a bone fracture. We elected to pursue the promising strategy of using small-molecule drug conjugates that deliver therapeutics to bone in an attempt to increase the efficacy and safety of drugs for treating bone-related diseases.</div><div>This strategy also opened the door for new methods of administering drugs. Traditionally, administering bone anabolic agents to treat bone fractures has relied entirely on local surgical application. However, because it is so invasive, this method’s use and development has been limited. By conjugating bone anabolic agents to bone-homing molecules, bone fracture treatment can be performed through minimally invasive subcutaneous administration. The exposure of raw hydroxyapatite that occurs with a bone fracture allows these high-affinity molecules to chelate the calcium component of hydroxyapatite and localize primarily to the fracture site.</div><div>Many bone-homing molecules (such as bisphosphonates and tetracycline targeting) have been developed to treat osteoporosis. However, many of these molecules have toxicity associated with them. We have found that short oligopeptides of acidic amino acids can localize to bone fractures with high selectivity and with very low toxicity compared to bisphosphonates and tetracyclines.</div><div>We have also demonstrated that these molecules can be used to target peptides of all chemical classes: hydrophobic, neutral, cationic, anionic, short, and long. This ability is particularly useful because many bone anabolics are peptidic in nature. We have found that acidic oligopeptides have better persistence at the site of the fracture than bisphosphonate-targeted therapeutics. This method allows for a systemic administration of bone anabolics to treat bone fractures, which it achieves by accumulating the bone anabolic at the fracture site. It also opens the door for a new way of treating the prevalent afflictions of broken bones and the deaths associated with them.</div><div>We further developed this technology by using it to deliver anabolic peptides derived from growth factors, angiogenic agents, neuropeptides, and extracellular matrix fragments. We found several promising therapeutics that accelerated the healing of bone fractures by improving the mineralization of the callus and improving the overall strength. We optimized the performance of these molecules by improving their stability, targeting ligands, linkers, dose, and dosing frequency.</div><div>We also found that these therapeutics could be used to accelerate bone fracture repair even in the presence of severe comorbidities (such as diabetes and osteoporosis) that typically slow the repair process. We found that, unlike the currently approved therapeutic for fracture healing (BMP2), our therapeutics improved functionality and reduced pain in addition to strengthening the bone. These optimized targeted bone anabolics were not only effective at healing bone fractures but they also demonstrated that they could be used to speed up spinal fusion. Additionally, we demonstrated that acidic oligopeptides have potential to be used to treat other bone diseases with damaged bone.</div><div>With these targeted therapeutics, we no longer have to limit bone fracture healing to casts or invasive surgeries. Rather, we can apply these promising therapeutics that can be administered non-invasively to augment existing orthopedic practices. As these therapeutics move into clinical development, we anticipate that they will be able to reduce the immobilization time that is the source of so many of the deadly complications associated with bone fracture healing, particularly in the elderly.</div>