The work presented in this thesis deals with the structural and functional properties of peptides at surfaces. The interaction of peptides with surfaces is an ever so common occurrence in our every day life, from the bug squashed on the windshield of our car to the barnacle on our boat, and from the blood plasma used in the hospital to the proteins in our cells. The effect these occurrences has on our lives is diverse, the bug is annoying whereas the barnacle settlement of ship hull is costly for marine transportation, the blood plasma contains components of vital importance for our immunological defense system and the proteins in our cells are crucial for regulatory processes and life.One part of this thesis, performed as a part of the EU-founded project AMBIO, deals with the concept of marine biofouling. A number of short peptides have been designed, synthesized, and used to investigate their effect on the settlement on marine biofoulers, such as the Ulva linza algae and the Navicula diatom, on template surfaces coated with thin layers of these molecules. The surfaces have been thoroughly investigated with respect of their physio-chemical properties before and after submersion in artificial seawater and ultimately in suspensions containing the organisms. The most interesting results were obtained with an arginine-rich peptide coating that when introduced to Ulva linza zoospores, displayed extensive settlement, compared to reference surfaces. In addition, a large fraction of the settled spores had an abnormal morphology.The other part of this thesis is focused on designed peptides that when adsorbed on a negatively charged surface adopts a well-defined secondary structure, either α-helical or β-sheet. Precisely placed amino acids in the peptides will strongly disfavor structure in solution, primarily due to electrostatic repulsion, but when the peptides are adsorbed on the negatively charged surfaces, they adopt a well-defined secondary structure due to ion pair bonding. These interactions have been thoroughly investigated by systematic variations of the side-chains. In order to determine the factors contributing to the induced structure, several peptides with different amino acid sequences have been synthesized. Factors that have been investigated include 1) the positive charge density, 2) distribution of positive charges, 3) negative charge density, 4) increasing hydrophobicity, and 5) incorporating amino acids with different helix propensities. Moreover, pH dependence and the effect of different interaction partners have also been investigated. It has also been shown that the system can be modified to incorporate a catalytic site that is only active when the helix is formed. This research will increase our understanding of peptide-surface interactions and might be of importance for both bionanotechnology and medicine.

<p>This thesis investigates three different surface modifications, and the route to design and synthesize them. The thesis is therefore divided into three sub- projects. (i.) Design and synthesis of a peptide which secondary structure could be controlled by a negatively charged surface. (ii.) Design and synthesis of a cyclic peptide, that would self-organize prior to surface interaction, using the type I anti-freeze protein of a winter flounder as template. (iii.) The use of solid-phase synthesis to make the synthesis of SAM-molecules easier.</p>

This thesis investigates three different surface modifications, and the route to design and synthesize them. The thesis is therefore divided into three sub- projects. (i.) Design and synthesis of a peptide which secondary structure could be controlled by a negatively charged surface. (ii.) Design and synthesis of a cyclic peptide, that would self-organize prior to surface interaction, using the type I anti-freeze protein of a winter flounder as template. (iii.) The use of solid-phase synthesis to make the synthesis of SAM-molecules easier.

No / Most rotamer libraries are generated from subsets of the PDB and do not fully represent the conformational scope of protein side chains. Previous attempts to rectify this sparse coverage of conformational space have involved application of weighting and smoothing functions. We resolve these limitations by using physics-based molecular dynamics simulations to determine more accurate frequencies of rotameric states. This work forms part of our Dynameomics initiative and uses a set of 807 proteins selected to represent 97% of known autonomous protein folds, thereby eliminating the bias toward common topologies found within the PDB. Our Dynameomics derived rotamer libraries encompass 4.8 × 10(9) rotamers, sampled from at least 51,000 occurrences of each of 93,642 residues. Here, we provide a backbone-dependent rotamer library, based on secondary structure ϕ/ψ regions, and an update to our 2011 backbone-independent library that addresses the doubling of our dataset since its original publication. / NIH

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

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-11-22T16:54:28Z No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2016-11-30T15:46:54Z (GMT) No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-30T15:46:54Z (GMT). No. of bitstreams: 2 Dissertação - Raisa Melo Lima - 2016.pdf: 5394977 bytes, checksum: 7066675bd31ee0e72d9ecd420ea0d1e7 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Brazil, where are recorded about 80% of cases worldwide, and has Paracoccidioides sp. as the etiologic agent. Malate synthase (MLS) is an important enzyme related to the fungal metabolism, once it is essential in the glyoxylate cycle, a secondary metabolic pathway of the citric acid cycle exclusive to microorganisms and plants. Its absence in humans makes this enzyme an interesting subject to study, mainly in rational drug design. From recent in vitro studies, several interacting proteins of MLS (receiver) were classified, but the modes of interaction and key regions involved in protein-protein interfaces (PPIs) have not yet been described. In this work, six (6) binding proteins (BPs) were selected to describe the IP's of MLS. Their tridimensonal structures, as well as MLS, were predicted by homology modeling using I-TASSER server, and subsequent molecular dynamics simulations (MD). The most common conformational modes of each protein were obtained by cluster analysis of the trajectories generated by MD. Molecular docking simulations using Gramm-X were then performed for the conformational modes of MLS against the BP's, resulting in a total of 36 complexes. Based on the higher frequency of some small fragments of proteins observed in the IPP's, 57 peptides with sizes between 5 and 20 residues, were initially selected from 5 regions of MLS which are considered more frequent in protein-protein interaction. FlexPepDock simulations were performed to optimize the atomic coordinates of the peptide complexed with MLS, and concomitantly, PepFOLD simulations were performed to evaluate the stability of each peptide in solution. Based on the lower energy score of peptides linked to MLS, and the stability of their structures in solution (MLS-free), 6 peptides were selected as promising ligands to MLS mode 1. The stability and patterns of interactions of these peptides were evaluated in detail. / A paracoccidioidomicose (PCM) é uma micose sistêmica endêmica no Brasil, onde são registrados cerca de 80% dos casos mundiais, e possui como agente etiológico o fungo Paracoccidioides spp. A Malato sintase (MLS) é uma importante enzima relacionada ao metabolismo fúngico, uma vez que é essencial no ciclo do glioxilato, uma via metabólica importante de produção de glicose para parede celular, sendo exclusiva de micro-organismos e plantas. Sua ausência em humanos a torna um alvo interessante de estudo, principalmente, no desenho racional de fármacos. A partir de recentes estudos in vitro, várias proteínas que interagem com a PbMLS (receptor) foram classificadas, porém os modos de interação e as regiões chaves envolvidas nas interfaces proteína-proteína (IPP’s), não foram ainda descritas. Neste trabalho, 6 (seis) proteínas ligantes (PL) foram selecionadas para verificar suas interações com PbMLS. As estruturas tridimensionais dessas proteínas, bem como de PbMLS, foram preditas por homologia, usando o servidor I-TASSER. Simulações de dinâmica molecular (DM) foram realizadas pelo programa GROMACS, e os modos das conformações mais representativas de cada proteína foram determinados baseando-se nas análises de agrupamentos a partir das trajetórias geradas por DM. Simulações de ancoragem molecular com GRAMM-X foram então realizadas entre os modos conformacionais de PbMLS contra os obtidos de PL’s, resultando num total de 36 complexos. Baseado na frequência maior de alguns pequenos fragmentos de proteínas, observados nas IPP’s, 57 peptídeos de tamanhos entre 5 e 20 resíduos de aminoácidos, foram inicialmente selecionados a partir de 5 regiões da PbMLS consideradas mais frequentes na interação proteína-proteína. Simulações com FlexPepDock foram realizadas para otimizar as coordenadas atômicas dos peptídeos complexados com MLS e, concomitantemente, simulações com PepFOLD foram realizadas para avaliar a estabilidade de cada peptídeo em solução. Com base nos mais baixos scores de energia dos peptídeos ligados a MLS bem como na estabilidade de suas estruturas não ligadas em solução , 5 peptídeos foram selecionados como promissores ligantes ao modo 1 de MLS. A estabilidade e os padrões de interações destes peptídeos são avaliados em detalhes.

[ES] La biogénesis ribosomal es uno de los procesos más complejos, esenciales y costosos energéticamente de la célula eucariota. La formación de las subunidades ribosomales en levaduras comienza en el nucléolo con la transcripción del ARNr. En este proceso se requiere la participación de más de doscientos factores de ensamblaje y ARNs pequeños nucleolares (snoARNs) que no formarán parte del ribosoma maduro aunque son necesarios para un apropiado procesamiento del ARNr y organización estructural de las subunidades ribosomales. Los estudios estructurales de los estadios de maduración de la subunidad 60S han permitido la identificación de algunas interacciones funcionales entre los distintos factores de ensamblaje. Entre ellos se encuentran las proteínas Nop7, Erb1 e Ytm1 que forman un heterotrímero discreto denominado subcomplejo Nop7 en levaduras, o complejo PeBoW en mamíferos, compuesto por los ortólogos Pes1, Bop1 y WDR12, respectivamente. Este complejo puede detectarse de forma aislada de las partículas prerribosomales. La formación de este heterotrímero es esencial para el ensamblaje de la subunidad 60S ya que garantizan la correcta maduración del extremo 5' del ARNr 5,8S facilitando así su asociación con el ARNr 25S, aunque se desconoce en detalle el papel exacto en la biogénesis ribosomal. En este trabajo se ha realizado un análisis de las interacciones entre los componentes del PeBoW así como una aproximación a la resolución estructural del complejo en solución. Usando una combinación de técnicas biofísicas, nuestros resultados indican que la conformación estructural que adopta el complejo PeBoW en el nucleoplasma es diferente a la descrita en el contexto prerribosomal. Además, se han identificado posibles funciones que podría estar ejerciendo el complejo PeBoW o bien las distintas proteínas del complejo de forma aislada fuera del prerribosoma. Asimismo se conoce que la biogénesis ribosomal es un mecanismo altamente regulado y estrechamente relacionado con crecimiento y proliferación celular. La imperiosa necesidad de síntesis de ribosomas y proteínas que tiene una célula tumoral convierte a este proceso en un punto débil de la misma. Por esta razón, hay un gran interés para estudiar la biogénesis ribosomal como diana terapéutica contra el cáncer. Se ha observado que la paralización de esta vía es capaz de promover la activación no genotóxica del supresor tumoral p53, a diferencia de los efectos indeseados que provocan las terapias convencionales contra el cáncer. Como primer paso hacia el desarrollo de herramientas inhibidoras de la biogénesis del ribosoma, hemos utilizado la información cristalográfica que poseíamos del complejo de Chaetomium thermophilum entre los factores de ensamblaje Erb1 e Ytm1 para realizar una selección guiada por la estructura de péptidos de interferencia. Los péptidos de interferencia han sido analizados in vitro para determinar su capacidad de interacción utilizando técnicas biofísicas. Además, se han generado péptidos de interferencia con la secuencia humana de Erb1/Ytm1 para evaluar sus efectos en cultivo de células de cáncer de colon HCT-116. Nuestros resultados indican que el estrés ribosómico se puede inducir en diferentes etapas del proceso de maduración al dirigirse a las interacciones proteína-proteína, elevándolas como una alternativa al uso de inhibidores de la ARN pol I. / [CA] La biogènesi ribosomal és un dels processos més complexos, essencials i costosos energèticament de la cèl·lula eucariota. La formació de les subunitats ribosomals en llevats comença en el nuclèol amb la transcripció de l'ARNr. En aquest procés es requereix la participació de més de dos-cents factors d'assemblatge i ARNs xicotets nucleolars (snoARNs) que no formaran part del ribosoma madur encara que són necessaris per a un apropiat processament de l'ARNr i organització estructural de les subunitats ribosomals. Els estudis estructurals dels estadis de maduració de la subunitat 60S han permès la identificació d'algunes interaccions funcionals entre els diferents factors d'assemblatge. Entre ells es troben les proteïnes Nop7, Erb1 i Ytm1 que formen un heterotrímer discret denominat subcomplex Nop7 en llevats, o complex PeBoW en mamífers, compost pels ortòlegs Pes1, Bop1 i WDR12, respectivament. Aquest complex pot detectar-se de forma aïllada de les partícules prerribosomals. La formació d'aquest heterotrímer és essencial per a l'assemblatge de la subunitat 60S ja que garanteix la correcta maduració de l'extrem 5' de l'ARNr 5,8S facilitant així la seua associació amb l'ARNr 25S, encara que es desconeix detalladament el paper exacte en la biogènesi ribosomal. En aquest treball s'ha realitzat una anàlisi de les interaccions entre els components del PeBoW així com una aproximació a la resolució estructural del complex en solució. Utilitzant una combinació de tècniques biofísiques, els nostres resultats indiquen que la conformació estructural que adopta el complex PeBoW en el nucleoplasma és diferent a la descrita en el context prerribosomal. A més, s'han identificat possibles funcions que podria estar exercint el complex PeBoW o bé les diferents proteïnes del complex de forma aïllada fora del prerribosoma. Així mateix es coneix que la biogènesi ribosomal és un mecanisme altament regulat i estretament relacionat amb creixement i proliferació cel·lular. La imperiosa necessitat de síntesi de ribosomes i proteïnes que té una cèl·lula tumoral converteix a aquest procés és un punt feble d'aquesta. Per aquesta raó, hi ha un gran interès per a estudiar la biogènesi ribosomal com a diana terapèutica contra el càncer. S'ha observat que la paralització d'aquesta via és capaç de promoure l'activació no genotòxica del supressor tumoral p53, a diferència dels efectes indesitjats que provoquen les teràpies convencionals contra el càncer. Com a primer pas cap al desenvolupament d'eines inhibidores de la biogènesi del ribosoma, hem utilitzat la informació cristal·logràfica que posseíem del complex de Chaetomium thermophilum entre els factors d'assemblatge Erb1 i Ytm1 per a realitzar una selecció guiada per l'estructura de pèptids d'interferència. Els pèptids d'interferència han sigut analitzats in vitro per a determinar la seua capacitat d'interacció utilitzant tècniques biofísiques. Així mateix, s'han generat pèptids d'interferència amb la seqüència humana de Erb1/Ytm1 per a avaluar els seus efectes en cultiu de cèl·lules de càncer de còlon HCT-116. Els nostres resultats indiquen que l'estrès ribosòmic es pot induir en diferents etapes del procés de maduració en dirigir-se a les interaccions proteïna-proteïna, elevant-les com una alternativa a l'ús d'inhibidors de l'ARN pol I. / [EN] Ribosomal biogenesis is a complex, essential and one of the most energy-costing process in eukaryotic cells. The assembling of the ribosomal subunits, in yeast, starts with the RNA transcription inside the nucleolus. This process requires the participation of more than two hundred assembly factors and small nucleolar RNAs (snoRNAs) that will not be part of the mature ribosome, however, they are still necessary to perform the appropriate rRNA processing and structural management. The structural studies on the maturation stages of the 60S subunit have shown functional interactions between different assembling factors. In this group, we find Nop7, Erb1 and Ytm1, that both form a discrete heterotrimer called Nop7 subcomplex in yeast, or PeBoW in mammalian, composed by the respective orthologues Pes1, Bop1 and WDR12. This complex can be found isolated from the pre-ribosomal particles. The presence of this complex is crucial to the 60S subunit assembling, once it ensures the correct maturation of the 5' tip of the rRNA 5,8S, supporting the association with the 25S rRNA. Yet, the details of the PeBoW complex roll in ribosomal biogenesis remain unknown. Hereafter, there are some analysis of interaction assays between the PeBoW component proteins, as well as an approach to structural of the complex in solution. Using different biophysical techniques, the results suggest that PeBoW complex adopts a different conformation in the nucleoplasm than the described in the pre-ribosomal context. Furthermore, it hints some other functions of the PeBoW complex, or the isolated component proteins out of the pre-ribosome path. Besides that, the ribosomal biogenesis is a highly regulated process and strictly related with cellular growth and proliferation. The mandatory demand of ribosome and protein synthesis of tumoral cells make this process a weak point of it. This is the reason why ribosomal biogenesis as cancer treatment target is a point of great interest. According to some studies, it is expected that the interruption of this pathway can lead the non-genotoxic activation of the tumor suppressor p53, unlike other conventional cancer therapies. As a first step to development of ribosomal biogenesis inhibiting tools, the structural information of Erb1 and Ytm1 in Chaetomium thermophilum complex, was taken to design structure-guided interfering peptides. Using biophysical techniques, the interfering peptides were evaluated in vitro to determine their interaction ability. Then, human-sequenced Erb1/Ytm1 interfering peptides were designed to look over the effects on colon cancer cells HCT-116. The results indicate that the ribosomal stress can be induced in different stages of the maturation process, by approaching protein-protein interactions, planting it as an alternative to RNA pol I inhibitors. / Este trabajo ha sido realizado con el apoyo económico de los proyectos de investigación enumerados a continuación: SAF2015-67077-R, SAF2017-89901-R y PROMETEO/2018/0 Durante el periodo de realización de esta tesis, la autora, Lidia Orea Ordóñez, ha sido beneficiaria de una subvención para la contratación de personal investigador de carácter predoctoral denominada “Ayudas para la contratación de personal investigador de carácter predoctoral”, (ACIF) (ACIF/2016/103), otorgada por la Generalitat Valenciana, Conselleria de Innovación, Universidades, Ciencia y Sociedad Digital, concedida en la convocatoria 2016. / Orea Ordóñez, L. (2022). Estructura y función del complejo PeBoW como modelo en el desarrollo de posibles herramientas terapéuticas [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/182347

Structural studies of peptides are of great importance in developing novel and effective biomaterials ranging from drugs and vaccines to nano materials with industrial applications. In addition, they provide model systems to study and mimic the protein conformations. The ability to generate folded intramolecularly hydrogen bonded structures in short peptides is essential for peptide design strategies, which rely on the use of folding nuclei in the construction of secondary structure modules like helices and β-hairpins. In these approaches, conformational choices at selected positions are biased, using local stereochemical constraints, that limit the range of accessible backbone torsion angles. X-ray crystallographic studies of designed peptides provide definitive proof of the success of a design strategy, and provide essential structural information that can be utilized in the future design of biologically and structurally important polypeptides. Recent trends in peptide research focus on the incorporation of β-, γ- and higher homologs of the α-amino acid residues in designed peptides as they confer more proteolytic stability to the polypeptides. X-ray crystallographic studies of such modified peptides containing non-protein residues are essential, since information on the geometric and stereochemical properties of modified amino acids can only be gathered from the systematic structural studies of synthetic peptides incorporating them. This thesis reports a systematic study of the structures and conformations of amino acid derivatives and designed peptides containing stereochemically constrained α-, β- and γ-amino acid residues and the structural studies of polymorphic peptide helices. The structures described in thesis contain the Cα,α-dialkyalted α-residues α-aminoisobutyric acid (Aib) and 1-aminocyclohexane-1-carboxylic acid (Ac6c), the β-amino acid residue 1-aminocyclohexane acetic acid (β3,3Ac6c) and the γ-amino acid residue 1-aminomethylcyclohexaneacetic acid (gabapentin, Gpn). The crystal structure determination of peptides incorporating conformationally constrained α-, β- and γ- amino acid residues permitted the characterization of new types of hydrogen bonded turns and polymorphs. The studies enabled the precise determination of conformational and geometric parameters of two ω-amino acid residues, gabapentin and β 3,3Ac6c and provided detailed information about the conformational excursions possible for peptide molecules. This thesis is divided into 10 chapters. Chapter 1 gives a general introduction to the stereochemistry of the polypeptide chain, description of backbone torsion angles of α- and ω- amino acid residues and the major secondary structures of α-peptides, β-peptides, γ-peptides and hybrid peptides. A brief introduction to polymorphism and weak interactions, in particular aromatic interactions, is also provided, followed by a discussion on X-ray diffraction and solution to the phase problem. Chapter 2 describes the crystal structures of gabapentin zwitterion and its eight derivatives (Ananda, Aravinda, Vasudev et al., 2003). The crystal structure of the gabapentin zwitterions determined in this study is identical to that previously reported (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643). Eight of the nine achiral compounds crystallized in centrosymmetric space groups P21/c, C2/c or Pbca, while one derivative (Tos-Gpn-OH) crystallized in non-centrosymmetric space group Pna21 with four independent molecules in the asymmetric unit.The structural studies presented in this chapter reveal that the geminal substituents on the Cβ atom limits the values of dihedral angles θ1 and θ2 to ±60°, resulting in folded backbone conformations in all the examples. Intramolecular hydrogen bonds with 7-atoms in the hydrogen bond turn (C7) are observed in three derivatives, gabapentin hydrochloride (GPNCL), Boc-Gpn-OH (BGPNH) and Piv-Gpn-OH (PIVGPN), while a 9-atom hydrogen bonded turn (C9) is observed in Ac-Gpn-OH (ACGPH). Unique structural features, such as an unusual anti conformation of the COOH group (in ACGPH) and positional disorder of the cyclohexane ring (in BGPNN), indicating the co-existence of both the interconvertible chair conformations, are revealed by the crystal structure analyses. Chapter 3 describes the structural characterization of novel hydrogen bonded conformations of homo oligomers of Gpn. The crystal structures of three peptides, Boc-Gpn-Gpn-NHMe (GPN2), Boc-Gpn-Gpn-Leu-OMe (GPN2L) and Boc-Gpn-Gpn-Gpn-Gpn-NHMe (GPN4) provide the first crystallographic characterization of two new families of polypeptide structures, the C9 helices and C9 ribbons (Vasudev et al., 2005, 2007), in which the molecular conformations are stabilized by contiguous C9 turns formed by the hydrogen bonding between the CO group of residue (i) and the NH group of residue (i+2). The C9 hydrogen bond is characterized by a specific combination of the four torsion angles for the Gpn backbone, with the torsion angles θ1 and θ2 adopting g+/g+ or g /g- conformations. The structural analysis also permits precise determination of hydrogen bond geometry for the C9 structures, which is highly linear in contrast to the analogous γ-turn hydrogen bonds in α-peptides. A comparison of the backbone conformations in the three peptides reveals two classes of C9 hydrogen bonded secondary structures, namely C9 helices and C9 ribbons. The packing arrangement in these γ-peptides follows the same patterns as the helix packing in crystals of α-peptides. Chapter 4 describes ten crystal structures of short hybrid peptides containing the Gpn residue (Vasudev et al., 2007). In addition to the C7 and C9 hydrogen bonded turns which are defined by the backbone conformations at the Gpn residue, hybrid turns defined by a combination of backbone conformations at the α and γ-residues or at the β and γ-residues have been determined. Peptides Boc-Ac6c-Gpn-OH (ACGPH), Piv-Pro-Gpn-Val-OMe (PPGPV) and Boc-Val-Pro-Gpn-OH (VPGPH) reveal molecular conformation stabilized by intramolecular C9 hydrogen bonds, while Boc-Ac6c-Gpn-OMe (ACGPO) and Boc-Gpn-Aib-OH (GPUH) are stabilized by a C7 hydrogen bonded turn at the Gpn residue. An αγ hybrid turn with 12 atoms in the intramolecular hydrogen bonded rings (C12 turns) has been observed in the tripeptide Boc-Ac6c-Gpn-Ac6c-OMe (ACGP3), while βγ hybrid turns with 13 atoms in the hydrogen bonded ring (C13 turns) have been characterized in the tripeptides Boc-βLeu-Gpn-Val-OMe (BLGPV) and Boc- βPhe-Gpn-Phe-OMe (BFGPF). The two βγ C13 turns belong to two different categories and are characterized by different sets of backbone torsion angles for the β and γ residues. A γα C10 hydrogen bond, which is formed in the N→C direction (NHi ••• COi+2), as opposed to the regular hydrogen bonded helices of α-peptides, has also been observed in BFGPF. The Chapter provides a comparison of the backbone torsion angles of the Gpn residue in various hydrogen bonded turns and a brief comparison of the observed hydrogen bonded turns with those of the α-peptides. Chapter 5 describes the crystal structures of three αγ hybrid peptides which show C12/C10 mixed hydrogen bond patterns (Vasudev et al., 2007, 2008a; Chatterjee, Vasudev et al.,2008a). The insertion of gabapentin in the predominantly α-amino acid sequences in Boc-Ala-Aib-Gpn-Aib-Ala-OMe (AUGP5) and Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe results in the observation of helices stabilized by αα C10 (310-turn) and αγ C12 turns. The tetrapeptide Boc-Leu-Gpn-Leu-Aib-OMe reveals a novel conformation, stabilized by C12 (αγ) and C10 (γα) hydrogen bonds of opposite hydrogen bond directionalities. The conformations observed in crystals have been extended to generate C12 helix and C12/C10 helix with alternating hydrogen bond polarities in ( αγ)n sequences. The structure determination of three crystals, providing five molecular conformations, presented in this chapter provides the first crystallographic characterization of two types of helices predicted for the regular αγ hybrid peptides from theoretical calculations. The crystal structure of Boc-Ala-Aib-Gpn-Aib-Ala-OMe also provides an example for the co-existence of left-handed and right-handed helix in the asymmetric unit. Chapter 6 describes the structural studies of αγ hybrid peptides containing Aib and Gpn residues, and is divided into two parts. The first part presents the crystal structure analysis of peptides of sequence length 2 to 4, with alternating Aib and Gpn residues, and illustrates the conformational variability in αγ hybrid sequences as evidenced by the observation of conformational polymorphs (Chatterjee, Vasudev et al., 2008b; Vasudev et al., 2007; Ananda, Vasudev et al., 2005). The peptide Boc-Gpn-Aib-NHMe (GUN), Boc-Aib-Gpn-Aib-OMe (UGU), Boc-Gpn-Aib-Gpn-Aib-OMe (GU4O), Boc-Aib-Gpn-Aib-Gpn-OMe (UG4O) and Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), all of which are potential candidates for exhibiting αγ C12 hydrogen bonds, reveal molecular conformations stabilized by diverse hydrogen bonded turns such as C7, C9, C12 and C17 in crystals. The conformational heterogeneity in this class of hybrid peptides is further evidenced by the observation of three polymorphs in the monoclinic space group P21/c for the tetrapeptide Boc-Aib-Gpn-Aib-Gpn-NHMe (UG4N), providing four independent peptide molecules adopting two distinct backbone conformations. In one polymorph, C12 helices terminated with an unusual three residue ( γαγ) C17 turn is observed, while the unfolding of helical conformation by solvent insertion into the backbone is observed in the other two polymorphs. The studies indicate the possible utility of Gpn residue in stabilizing locally folded conformations in the folding pathway, thus permitting their crystallographic characterization in multiple crystal forms. A discussion of the structural and conformational features of Gpn residues determined from all the crystal structures is presented in the Chapter, along with a φ-ψ plot for the Gpn residue. Part 2 of Chapter 6 describes the crystal structures of two octapeptides, Boc-Gpn-Aib-Gpn-Aib-Gpn-Aib-Gpn-Aib-OMe (GU8) and Boc-Leu-Phe-Val-Aib-Gpn-Leu-Phe-Val-OMe (LFVUG8), featuring C12 turns at the Aib-Gpn segments (Chatterjee, Vasudev et al., 2009). GU8 folds into a C12 helix flanked by C9 hydrogen bonds at both the termini, while LFVUG8 adopts β-hairpin conformation with a chain-reversing C12 turn at the central Aib-Gpn segment. A remarkable feature of the Aib-Gpn turn in the β-hairpin structure is the anti conformation about the Cβ-Cα (θ2) bond, which is the only example of a Gpn residue not adopting gauche conformation for both θ1 and θ2. The crystal structures of the two peptides, mimicking the two major secondary structural elements of α-peptides in hybrid polypeptides, permits a comparative study of the mode of molecular packing in crystals of α-peptides and hybrid peptides. The chapter also discusses theoretical calculations on αγ hybrid sequences, which reveal new types of C12 hydrogen bonded turns. Chapter 7 describes the crystal structures of conformationally biased tert-butyl derivatives of Gpn. The crystallographic characterization of the E (trans) and Z (cis) isomers of the residue,three protected derivatives and a tripeptide provides examples of C7 and C9 hydrogen bonded conformations, suggesting that the C7 and C9 hydrogen bonds can be formed by Gpn residues with both the chair conformations of the cyclohexane ring. Chapter 8 describes the systematic structural studies of the derivatives and peptides of the stereochemically constrained β- amino acid residue, β3,3Ac6c (Vasudev et al., 2008c). The backbone torsion angles φ and θ adopt gauche conformation in majority of the examples, owing to the presence of a cyclohexane ring on the Cβ atom. In contrast to Gpn, β3,3Ac6c does not show strong preference for adopting intramolecularly hydrogen bonded conformations. Of the 16 crystal structures determined, intramolecular hydrogen bonds involving the β-residue are observed only in 4 cases. The amino acid zwitterion (BAC6C), the hydrochloride (BACHCL) and the dipeptide Boc-β3,3Ac6c-β3,3Ac6c-NHMe (BAC62N) form N-H•••O hydrogen bonds with 6-atoms in the hydrogen bond ring (C6 turns). An αβ hybrid C11 hydrogen bonded turn is characterized in the dipeptide Piv-Pro-β3,3Ac6c-NHMe, which is distinctly different from the C11 hydrogen bonds observed in αβ hybrid peptide helices. Several unique structural features such as a dynamic disorder of the hydrogen atom of the carboxylic acid group (in BBAC) and cis geometry of the urethane bond (in BBAC, BAC62N and BPBAC) have been observed in this study. A comparison of the backbone conformations of β3,3Ac6c with other β- amino acid residues is also provided. Chapter 9 describes the crystallographic characterization of a new polymorph of gabapentin monohydrate and crystal structures of the zwitterions of E and Z isomers of tert-butylgabapentin and its hydrochloride and hydrobromide (Vasudev et al., 2009). A comparison of the crystal structures of the monoclinic form (Ibers, J. A. Acta Crystallogr. 2001, C57, 641-643) of gabapentin monohydrate and the newly characterized orthorhombic form reveals identical molecular conformations and intermolecular hydrogen bond patterns in both the polymorphs. The two polymorphs show differences in the orientation of molecules constituting a layer of hydrophobic interactions between the cyclohexyl side chains. A comparison of the packing arrangements of the zwitterionic amino acid molecules in the crystal structures of gabapentin monohydrate, the tert-butyl derivatives and other co-crystals of gabapentin that had been characterized so far, is provided which would facilitate prediction of new polymorphs of the widely used drug molecule, Gpn. Chapter 10 describes the crystallization of α-peptide helices in multiple crystal forms (Vasudev et al., 2008b). Crystal structures of two peptides, Boc-Leu-Aib-Phe-Phe-Leu-Aib-Ala-Ala-Leu-Aib-OMe (LFF), Boc-Leu-Aib-Phe-Ala-Leu-Ala-Leu-Aib-OMe (D1) in two crystal forms and the crystal structure of a related sequence, Boc-Leu-Aib-Phe-Ala-Phe-Aib-Leu-Ala-Leu-Aib-OMe (D10) permit an analysis of the molecular conformation and packing patterns of peptide helices in crystals. The two polymorphs of LFF, crystallized in the space groups P21 and P22121, reveal very similar molecular conformation (α/310-helix) in both the polymorphic crystals; the two forms differ significantly in the pattern of solvation. The crystal structure determination of a monoclinic (P21) and an orthorhombic polymorph (P21212) of D1 provides five different peptide conformations, four of which are α-helical and one is a mixed 310/α-helix. The crystal structure determination of the three peptides provide an opportunity to compare the nature and role of aromatic interactions in stabilizing molecular conformation and packing and its significance in the observation of polymorphism. An analysis of the Cambridge Structural Database and a model for nucleation of crystals in hydrophobic peptide helices are also discussed.

No description available.

No description available.