Single-molecule localisation microscopy (SMLM), has allowed for optical microscopy to probe biological systems beyond the diffraction limit. The intrinsic 3D nature of biology has motivated the development of 3D-SMLM with novel techniques, including the double-helix point spread function (DHPSF). A bespoke microscope platform employing the DHPSF transformation was built, achieving ~10 nm lateral and ~20 nm axial localisation precision over a ~4 μm axial depth. Until recently, the DHPSF has been limited by spherical aberration present when imaging away from coverslip surfaces to the study of small volumes close to the coverslip. By matching the refractive index of the objective lens immersion liquid to that of the imaging media, this aberration can be minimised, facilitating large-volume imaging away from unphysiological flat surfaces. The work presented in this thesis illustrates the capabilities of the DHPSF for 3D-SMLM and single-particle tracking (SPT) in previously inaccessible areas of biological samples (e.g. in the nucleus and on the apical cell surface). Application of the DHPSF for SPT in eukaryotic cells are presented; tracking the motion of T-cell membrane proteins on the apical surface and components of the chromosome remodelling complex in the nucleus of embryonic stem cells. For these applications, meansquared displacement and jump distance diffusion analysis methodologies were extended into 3D and benchmarked against simulated datasets. A variety imaging applications that are facilitated by the extended depth of focus of the DHPSF are presented, focusing on quantification of T-cell membrane protein reorganisation upon immunological activation. Finally, the clustering distribution of the T-cell receptor is investigated by Ripley’s K analysis enabled by duel labelling of its position and the outer membrane in primary T cells.

O objetivo do presente estudo foi avaliar a atividade eletromiográfica dos músculos masseter e temporal e o comportamento esquelético e dental de crianças submetidas à expansão maxilar com o aparelho quadri-hélice. A amostra foi composta por doze crianças (10 meninas e 2 meninos), com idade média de 7 anos e 4 meses, portadoras de mordida cruzada posterior unilateral. Foram realizados traçados cefalométricos laterais antes do início do tratamento (T1) e após a remoção do aparelho (T2). A atividade eletromiográfica dos músculos masseter e temporal foi analisada nas situações clínicas de repouso muscular, apertamento dental máximo e mastigação não habitual e habitual, antes do início do tratamento (T1) e um mês após a remoção do aparelho quadri-hélice (T2). As medidas cefalométricas e eletromiográficas foram submetidas à análise estatística utilizando os programas GraphPad Prism e SPSS for Windows, respectivamente. A diferença das médias T1 e T2 foi avaliada pelo teste-t para medidas repetidas. Os resultados da análise cefalométrica mostraram que a expansão maxilar com o aparelho quadri-hélice não promoveu alterações esqueléticas ântero-posteriores e verticais significantes. Apenas a medida 1-PP apresentou aumento significante dentre as avaliadas no padrão dental. Na condição clínica de repouso, a análise eletromiográfica indicou uma diminuição na atividade do músculo masseter e um aumento significante na atividade do temporal. Durante o apertamento dental, a atividade eletromiográfica dos músculos masseteres apresentou uma leve diminuição e dos temporais manteve-se constante ao final do tratamento. Todos os músculos apresentaram aumento na atividade eletromiográfica durante a mastigação não habitual, sendo estatisticamente significante apenas para o músculo temporal direito. A atividade eletromiográfica diminuiu significantemente em todos os músculos avaliados na condição clínica de mastigação habitual após a remoção do aparelho quadri-hélice. / The aim of the present study was to analyze the electromyographic activity of masseter and temporal muscles and the skeletal and dental effects of maxillary expansion realized in children with the quad-helix appliance. The sample consisted of twelve children (10 girls and 2 boys), mean age 7 years and 4 months, with unilateral posterior crossbite. Lateral cephalograms were taken before treatment (T1) and after the quad-helix appliance was removed (T2). The electromyographic activity of masseter and temporal muscles was analyzed before treatment (T1) and after the appliance was removed (T2). The muscular activity was electromyographic analyzed during the clinical situation of rest, maximal voluntary dental clench, non-habitual and habitual chewing. The cephalometric and electromyographic measurements were analyzed statiscally using GraphPad Prism and SPSS 10.0 for Windows, respectively. The differences between T1 and T2 data were evaluated using the paired t- test. The results of the cephalometric analyze showed that the maxillary expansion realized in children with the quad-helix appliance didn?t promote vertical and sagittal skeletal significant change. Only the linear measure 1-PP showed a significant raise considering the dental pattern. During the clinical situation of rest, the electromyographic analyze indicated a diminution on the activity of the masseter muscle and a significant increase on the temporal activity. During maximal voluntary clench, the electromyographic activity of masseter muscle presented a slight diminution and the temporal activity stayed the same at the end of the treatment. Every muscle showed a raise on the electromyographic activity during non-habitual chewing, being statistically significant only for right temporal muscle. The electromyographic activity diminished significantly for every muscle evaluated during the clinical situation of habitual chewing after removal of the quad-helix appliance.

Submitted by Joana Azevedo (joanad@id.uff.br) on 2017-08-08T14:33:15Z No. of bitstreams: 1 Dissert Isabella Stroppa Rodrigues.pdf: 1327591 bytes, checksum: fc98fb32d877001047045439792c4e06 (MD5) / Approved for entry into archive by Biblioteca da Escola de Engenharia (bee@ndc.uff.br) on 2017-09-15T16:46:06Z (GMT) No. of bitstreams: 1 Dissert Isabella Stroppa Rodrigues.pdf: 1327591 bytes, checksum: fc98fb32d877001047045439792c4e06 (MD5) / Made available in DSpace on 2017-09-15T16:46:06Z (GMT). No. of bitstreams: 1 Dissert Isabella Stroppa Rodrigues.pdf: 1327591 bytes, checksum: fc98fb32d877001047045439792c4e06 (MD5) Previous issue date: 2016-12-15 / Esta pesquisa visou contribuir para a compreensão da complexidade de fatores envolvidos no relacionamento entre a universidade e outros entes, sendo, neste caso específico, o foco direcionado para as empresas e o governo, a partir das perspectivas da Tripla Hélice e da Universidade Empreendedora. A universidade brasileira, inserida desde sua criação em um contexto de constantes modificações e reformas, passa constantemente pelo questionamento acerca do seu papel. Recebendo inicialmente a atribuição de ser formadora de mão de obra, inquietações começam a surgir no sentido de enriquecer a atuação da universidade com a possibilidade de formação crítica dos indivíduos e criação de conhecimentos inovadores. Este conflito de percepções perpassa décadas e chega aos dias atuais inserido em um cenário onde se vislumbra o desinvestimento no ensino público brasileiro, o que torna atrativo para as universidades trilhar o caminho de formação de profissionais de acordo com o perfil requisitado pelo mercado, bem como direcionar as pesquisas da universidade para as necessidades das empresas devido à possibilidade de serem elas as potenciais financiadoras das atividades da universidade. No entanto, esta concepção não é linear e nem homogênea no cenário universitário em geral, e também não o é no contexto que envolve a Universidade Federal de Juiz de Fora, onde se aplicou a presente pesquisa utilizando o método do estudo de caso. O intuito desta pesquisa foi compreender como a Universidade Federal de Juiz de Fora se relaciona com a indústria e o governo e, ao mesmo tempo, identificar se ela está demonstrando possuir as características prescritas pelo modelo de Universidade Empreendedora. Neste sentido, foram analisados os três entes envolvidos a partir do estudo de seus posicionamentos oficiais e também, no caso da indústria e da universidade, de entrevistas realizadas com atores institucionais. Este estudo se balizou pela busca da produção de conhecimento capaz de contribuir para o enriquecimento da compreensão acerca desse complexo fenômeno de aproximação da esfera pública com a iniciativa privada, não se limitando apenas a descrições, mas também a explorar as problemáticas decorrentes do tema. / Entrepreneurial University. The Brazilian university, inserted since its creation in a context of constant changes and reforms, constantly goes through the questioning about its role. Initially receiving the assignment of being a trainer of labor, concerns begin to emerge in order to enrich the performance of the university with the possibility of critical formation of individuals and creation of innovative knowledge. This conflict of perceptions goes through decades and reaches the current days inserted in a scenario where the disinvestment in the Brazilian public education is envisaged, which makes it attractive for universities to follow the path of professional training according to the profile required by the market, as well as to direct university research to the needs of companies due to the possibility that they are the potential financiers of university activities. However, this conception is neither linear nor homogeneous in the university scenario in general, nor is it in the context involving the Federal University of Juiz de Fora, where the present research was applied using the case study method. The purpose of this research was to understand how the Federal University of Juiz de Fora relates to industry and government and, at the same time, to identify if it is demonstrating the characteristics prescribed by the Entrepreneurial University model. In this sense, the three entities involved in the study were analyzed from their official positions and also, in the case of industry and university, from interviews with institutional actors. This study was based on the search for the production of knowledge capable of contributing to the enrichment of the understanding about this complex phenomenon of approaching the public sphere with the private initiative, not only being limited to descriptions, but also exploring the problems arising from the theme.

Receptores nucleares formam uma superfamília de proteínas responsáveis pela regulação da expressão de genes. Estruturalmente, são formados por três domínios: um domínio N-terminal bastante variável, um domínio altamente conservado de ligação com o DNA e um domínio C-terminal, menos conservado, denominado domínio de ligação com o ligante (LDB). Diversos experimentos mostram que a interação com o ligante afeta a estrutura e a mobilidade da hélice C-terminal dos receptores nucleares (hélice 12 do domínio de ligação com o ligante), sendo o principal mecanismo de ativação e repressão da transcrição. As primeiras estruturas de LBDs de receptores nucleares revelaram importantes diferenças entre estruturas contendo ligantes (holo) e estruturas apo, principalmente no que diz respeito a posição da hélice 12: em estruturas apo, foi observada a H12 em uma conformação aberta, expondo o sítio de ligação com o ligante, enquanto que em estruturas holo, foi observada a H12 em uma conformação fechada, dobrada sobre o corpo do LBD e envolvendo completamente o ligante. Essa diferença sugeriu um mecanismo para a entrada e saída de ligantes do sítio de ligação denominado modelo da ratoeira, entretanto, esse modelo apresenta diversas inconsistências e tem sido desacreditado. Estudos experimentais e teóricos recentes mostram que a hélice 12 é mais móvel na ausência de ligantes, entretanto, esses estudos não fornecem evidencias de que o aumento da mobilidade da está associado com o deslocamento da H12 em relação ao corpo do LBD, como sugerido pelo modelo da ratoeira. Embora esteja claro que a hélice 12 é mais móvel na ausência de ligantes, a dimensão da variação conformacional sofrida pela hélice 12 ainda não está clara. Nesse trabalho buscamos a construção de um modelo capaz de dimensionar a mobilidade da hélice 12 através da comparação direta entre simulações de dinâmica molecular e experimentos de anisotropia de fluorescência resolvida no tempo. Utilizando simulações de dinâmica molecular reproduzimos experimentos de anisotropia de fluorescência acoplando a sonda cys-flúor a hélice 12 do PPARγ para estudar sua mobilidade. Mostramos que as observações experimentais só podem ser explicadas por conformações onde a sonda fluorescente permanece presa a superfície do LBD. Foi mostrado também que curvas de anisotropia com decaimentos comparáveis com os decaimentos experimentais estão associados a pequenas variações conformacionais de hélice 12. Simulações para dois modelos de apo-PPARγ com a H12 aberta em relação ao corpo do LBD e para as estruturas cristalográficas de apo-RXR e apo-ER, onde a H12 também adota uma conformação aberta, revelaram curvas de anisotropia com decaimentos mais rápidos que os experimentais. Esses resultados implicam em um modelo onde a H12 sofre alterações conformacionais locais, não apresentando variações tão dramáticas como o proposto pelo modelo da ratoeira. / Nuclear Hormone Receptors comprise a protein superfamily responsible for regulation of gene expression. Structurally, they are composed by three domains: a variable N-terminal domain, a highly conserved DNA-binding domain (DBD), and a less conserved C-terminal domain, known as ligand binding domain (LBD). Many experiments have shown that the interaction with ligands affects the structure and the mobility of nuclear receptors C-terminal helix (LBDs Helix 12), being the main mechanism of transcription activation and repression. The first nuclear receptor LBDs structures revealed important differences between ligand bound (holo) and apo-structures concerning the position of the H12: in apo structures, H12 adopted an open conformation, exposing the ligand binding pocket, whereas in holo structures, the H12 was closed, packed over the body of the LBD, burying completely the ligand. This difference suggested a mechanism for ligand entry and exit from the binding pocket called mouse-trap model, however this model has several inconsistencies and has been discredited. Recent experimental and theoretical studies have shown that H12 is more labile in the absence of ligand, but these studies dont provide evidences that the increase in the mobility is associated with the detachment of H12 from the body of the LBD as suggested by the mouse-trap model. Although its clear that H12 is more flexible in the absence of ligands, the size of the conformational changes undergone by H12 is not yet clear. In this work we seek to construct a definitive model for the range of motions that H12 may undergo in the presence or absence of ligand using molecular dynamics simulations. Through direct comparison between molecular dynamics simulations and time-resolved fluorescence anisotropy experiments, we show that experimental observation can only be explained by conformations where the fluorescent probe is interacting with the surface of the PPARγ surface. We also show that simulations with anisotropy decay rates comparable to the experimental decay are associated with small helix 12 conformational changes. Simulations with two models of apo-PPARγ with H12 detached from the body of the LBD and with crystallographic structures of apo-RXR and apo-ER, where the H12 also is in an open conformation, display anisotropy decay rates significantly faster than the experimental ones. These results imply a model for the molecular mobility of the LBD where H12 undergoes local conformational changes and should exhibit dynamic properties less dramatic than proposed by the mouse trap model.

<p>Structural bioinformatics deals with the analysis, classification and prediction of three-dimensional structures of biomacromolecules. It is becoming increasingly important as the number of structures is growing rapidly. This thesis describes three studies concerned with protein-function prediction and two studies about protein structure validation.</p><p>New protein structures are often compared to known structures to find out if they have a known fold, which may provide hints about their function. The functionality and performance of eleven fold-comparison servers were evaluated. None of the tested servers achieved perfect recall, so in practise a combination of servers should be used.</p><p>If fold comparison does not provide any hints about the function of a protein, structural motif searches can be employed. A survey of left-handed helices in known protein structures was carried out. The results show that left-handed helices are rare motifs, but most of them occur in active or ligand-binding sites. Their identification can therefore help to pinpoint potentially important residues.</p><p>Sometimes all available methods fail to provide hints about the function of a protein. Therefore, the potential of using docking techniques to predict which ligands are likely to bind to a particular protein has been investigated. Initial results show that it will be difficult to build a reliable automated docking protocol that will suit all proteins.</p><p>The effect of various phenomena on the precision of accessible surface area calculations was also investigated. The results suggest that it is prudent to report such values with a precision of 50 to 100 Ų.</p><p>Finally, a survey of register shifts in known protein structures was carried out. The identified potential register shifts were analysed and classified. A machine-learning approach ("rough sets") was used in an attempt to diagnose register errors in structures.</p>

<p>This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of <i>p</i>-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of <i>bis</i>(neopentyl glycolato)diboron or <i>bis</i>(pinacolato)diboron and 2-I-<i>p</i>-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules.</p><p>The DNA intercalator doxorubicin has been functionalized to enable ¹²⁵I labelling. The aim of combining the DNA intercalator with ¹²⁵I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation.</p><p>The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.</p>

This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of p-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of bis(neopentyl glycolato)diboron or bis(pinacolato)diboron and 2-I-p-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules. The DNA intercalator doxorubicin has been functionalized to enable 125I labelling. The aim of combining the DNA intercalator with 125I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation. The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.

Structural bioinformatics deals with the analysis, classification and prediction of three-dimensional structures of biomacromolecules. It is becoming increasingly important as the number of structures is growing rapidly. This thesis describes three studies concerned with protein-function prediction and two studies about protein structure validation. New protein structures are often compared to known structures to find out if they have a known fold, which may provide hints about their function. The functionality and performance of eleven fold-comparison servers were evaluated. None of the tested servers achieved perfect recall, so in practise a combination of servers should be used. If fold comparison does not provide any hints about the function of a protein, structural motif searches can be employed. A survey of left-handed helices in known protein structures was carried out. The results show that left-handed helices are rare motifs, but most of them occur in active or ligand-binding sites. Their identification can therefore help to pinpoint potentially important residues. Sometimes all available methods fail to provide hints about the function of a protein. Therefore, the potential of using docking techniques to predict which ligands are likely to bind to a particular protein has been investigated. Initial results show that it will be difficult to build a reliable automated docking protocol that will suit all proteins. The effect of various phenomena on the precision of accessible surface area calculations was also investigated. The results suggest that it is prudent to report such values with a precision of 50 to 100 Å2. Finally, a survey of register shifts in known protein structures was carried out. The identified potential register shifts were analysed and classified. A machine-learning approach ("rough sets") was used in an attempt to diagnose register errors in structures.

Proteins performing catalytic roles predominantly occur in a few protein folds. Functional diversity within a common structural scaffold has been attributed to conformational features that enable exploration of reaction space. In this study, we examined specific aspects of functional diversity in the Double Stranded β-helix(cupin) fold. The cupin domain is a hyper-stable protein fold that can support a variety of functions. Variation in function using a conserved active site in the cupin fold is achieved by changes in the residues that line the active site cavity as well as by the choice of a metal cofactor. Although this appears to be a likely basis for functional diversification, a few exceptions exist. It is thus interesting to examine how enzymes with the same structure, metal cofactor and ligand coordination catalyze a diverse range of reactions. This thesis describes two bi-cupins, BacB (also known as bacilysin synthase, YwfC) and Quercetinase (YxaG). BacB is a part of the protein machinery involved in the synthesis of a di-peptide antibiotic bacilysin. The case of the bicupin protein BacB illustrates the problem of functional annotation of proteins with the cupin fold. None of the predicted functions for this enzyme could be experimentally validated in vitro. The crystal structure, determined by Single-wavelength Anomalous Dispersion (SAD) based on the bound metal-ion at the active site provided a basis to evaluate the catalytic role of this protein. Eventually, the function of this protein could be determined based on characterizing the gene product of bacA, the gene preceding bacB in the B. subtilis bac operon. The crystal structure determination of BacB also led to an analysis of multiple crystal forms, with implications for the role of molecular symmetry in forming protein crystals. The stability of the cupin domain was examined using B. subtilis quercetinase as a model system. The availability of the crystal structure and a robust activity assay enabled us to examine the role of fragment complementation in the stability of the cupin scaffold and its implications for the function of this enzyme. This thesis also has a section on the use of structural homology for function annotation for cupin proteins. The results presented here thus provide a frame-work to understand the structural basis for functional diversity in the cupin family. This thesis is organized as follows: Chapter 1: This chapter provides an introduction to the Double Stranded β-Helix-Helix (DSBH or cupin) fold. Proteins with a cupin scaffold are remarkably diverse - spanning both enzymatic and non-enzymatic functions. This chapter presents a compilation of previous reports encompassing eighteen different functional classes. These functions include seed storage, transcription factors and a host of various enzymatic activities. Cupin proteins can be monocupins, bicupins or multi-domain cupins based on the number of DSBH domains in a single polypeptide chain. Very few multi-domain cupin proteins have been identified and this is generally not considered to be a significant sub-group. The inference that cupin proteins with more than one domain are products of gene duplication events is also examined in detail. The latter part of this chapter aims to provide an introduction to the two model proteins B. subtilis BacB and Quercetinase. Chapter 2: This chapter describes studies on a bi-cupin protein BacB involved in bacilysin synthesis. Bacilysin is a non-ribosomally synthesized dipeptide antibiotic that is active against a wide range of bacteria and some fungi. Synthesis of bacilysin (L-alanine-[2,3-epoxycyclohexano-4]-L-alanine) is achieved by proteins in the bac operon, also referred to as the bacABCDE (ywfBCDEF) gene cluster in B. subtilis. The production of this antibiotic is regulated via a stringent response and branches off the pathway for aromatic amino-acid biosynthesis at prephenate. Extensive genetic analysis from several strains of B. subtilis suggests that the bacABC gene cluster encodes all the proteins that synthesize the epoxyhexanone ring of L-anticapsin. This data, however, could not be reconciled with the putative functional assignments for these proteins whereby BacA, a prephenate hydratase along with a potential isomerase/guanylyl transferase, BacB and an oxidoreductase, BacC, could synthesize L-anticapsin. Here, based on the characterization of the reaction products of BacA and BacB as well as the crystal structure of BacB, we demonstrate that B. subtilis BacB catalyzes the synthesis of 2-oxo-3-(4-oxocyclohexa-2,5-dienyl)propanoic acid, a precursor to L-anticapsin. The mass and NMR spectra of the reaction product of BacA suggest that BacA is a decarboxylase that acts on prephenate. BacB is an oxidase. This protein is a bi-cupin, with two putative active sites each containing a bound metal ion. Additional electron density at the active site of the C-terminal domain of BacB could be interpreted as a bound phenylpyruvicacid (PPY). A significant decrease in the catalytic activity of a point variant of BacB with a mutation at the N-terminal domain suggests that the N-terminal cupin domain is involved in catalysis. Chapter 3 is based on the crystal packing analysis of three different crystal forms of B. subtilis BacB. BacB is an oxidase that catalyzes the production of the di-peptide antibiotic bacilysin. This protein is a bi-cupin with two double stranded β-helix domains fused in a compact arrangement. BacB crystallizes in three crystal forms, belonging to the triclinic, monoclinic and tetragonal space groups. These different crystal forms could be obtained in similar crystallization conditions. We also note that a slight disturbance to the crystallization droplet results in nucleation events, eventually resulting in a different crystal form. All three crystal forms of BacB diffract to high resolution, thus enabling the structure determination and analysis of the packing arrangements of BacB in different space groups. Metal ions at the lattice interface dominate the different packing arrangements. The crystal packing reveals that a dimer of BacB serves as the template on which higher order symmetrical arrangements are formed. BacB, however, is a monomer in solution. The different crystal forms of BacB thus provide experimental evidence to the hypothesis that molecular symmetry could aid crystallization. Chapter 4 provides a conformational analysis of the cupin fold using B. subtilis quercetinase as a model system to understand the conformational determinants of functional diversity. Controlled proteolysis experiments revealed that this enzyme is active, thermo-stable and maintains its quaternary arrangement even after substantial (ca 33 %) cleavage of the protein. The results presented in this chapter thus show that the cupin scaffold offers a balance between protein stability and function by locating the active site and substrate recognition features in the most stable region of the protein. Chapter 5 is based on the phylogenetic analysis of cupin domains. The members of cupin superfamily exhibit large variations in their sequences, functions, organization of domains, quaternary association and the nature of bound metal ion despite having a conserved β-barrel structural scaffold. Here, an attempt was made to understand structure-function relationships among the members of this diverse superfamily and identify the principles governing functional diversity. The cupin superfamily also contains proteins for which structures are available through world-wide structural genomics initiatives but characterized as “hypothetical”. We have explored the feasibility of obtaining clues to functions of such proteins by means of comparative analysis with cupins of known structure and function. This phylogenetic strategy was applied to BacB leading to clustering with oxidoreductases. BacB was experimentally demonstrated to be an oxidase. Chapter 6 is a summary of the work reported in this thesis and the conclusions that can be drawn based on these studies. The appendix section of this thesis comprises additional experimental details, methodology and aspects of the techniques used in this study. Appendix I contains a description of a methodology for Molecular Replacement (MR) calculations in obtaining phase information for protein crystallography. Appendix II provides additional details of experimental protocols.

Replicating the multi-hierarchical self-assembly of collagen (peptide chain to triple helix to nanofiber and, finally, to a hydrogel) has long attracted scientists, both from the fundamental science perspective of supramolecular chemistry and for the potential biomedical applications perceived in tissue engineering. In terms of triple helical formation, collagen is the most abundant protein in the human body with at least 28 types, yet research involving collagen mimetic systems has only recently began to consider the innate ability of collagen to control helix composition and register. Collagen triple helices can be homotrimeric or heterotrimeric and while some types of natural collagen form only one specific composition of helix, others can form multiple. It is critical to fully understand and, if possible, reproduce the control that native collagen has on helix composition and register. In terms of nanofiber formation, many approaches to drive the self-assembly of synthetic systems through the same steps as natural collagen have been partially successful, but none have simultaneously demonstrated all levels of structural assembly. In this work, advancements in the ability to control helix composition and replicate the multi-hierarchical assembly of collagen are described. Both positive and negative design for the assembly of AAB type collagen heterotrimers were utilized by promoting heterotrimer formation though the use of charged amino acids to form intra-helix electrostatic interactions, while simultaneously discouraging homotrimers, resulting in the identification of multiple peptide systems with full control over the composition of the resulting triple helix. Similar salt-bridged hydrogen bonds between charged residues were incorporated into nanofiber forming peptides, one of which successfully assembled into sticky-ended triple helices, nanofibers with characteristic triple helical packing visible in the solution state, and strong hydrogels that are degraded by collagenase at a similar rate to natural collagen. Together, these results provide a better understanding of the self-assembly of collagenous sequences as well as a novel design scheme for synthetic extracellular matrix mimetics with potential applications in regenerative medicine and drug delivery.