Orientadores: Eduardo Cardoso de Abreu, Carlile Campos Lavor / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Matemática Estatística e Computação Científica / Made available in DSpace on 2018-08-22T08:30:13Z (GMT). No. of bitstreams: 1 Maioli_DouglasSilva_M.pdf: 4390735 bytes, checksum: 3008997a23c0723a152e019324a6f6a2 (MD5) Previous issue date: 2013 / Resumo: No presente trabalho é abordado um Problema de Geometria de Distâncias Moleculares (PGDM) que consiste na determinação de estruturas tridimensionais de moléculas a partir de distâncias entre pares de seus átomos. Inicialmente, apresentamos métodos da literatura utilizados para tentar resolver tal problema, como o Updated Geometric Build-Up (UGB) de Wu e Wu (2007) e o Algoritmo T (AT) de Fidalgo (2011). O novo método introduzido nesta dissertação de mestrado é baseado no AT e foi denominado de Algoritmo T Atualizado (ATA). Esta nova proposta utiliza a mesma estratégia desenvolvida no UGB, que busca obter uma maior estabilidade, com respeito ao número de condição, dos sistemas lineares resolvidos na execução do ATA. Por fim, um estudo baseado em experimentos numéricos foi feito para a verificação da qualidade das soluções obtidas pelo ATA, levando em conta o custo computacional, e em comparação com o método UGB / Abstract: The present work approaches the Molecular Distance Geometry Problem (MDGP) which consists on determining three-dimensional molecular structures from distance values between pairs of its atoms. Initially, we present methods from the literature which have been used in order to solve this problem, such as the Updated Geometric Build-Up (UGB) algorithm, from Wu and Wu (2007), and the T Algorithm (TA), from Fidalgo (2011). The new method, introduced in this master dissertation, is based on the TA and was named Updated T Algorithm (UTA). This new approach uses the same strategy developed in the UGB, which looks for obtaining a better numerical stability, with respect to the condition number of the coefficient matrices of the linear systems which are solved in UTA. Finally, a study based on numerical experiments was done for verifying the quality of the solutions obtained from UTA, considering the computational cost and comparing with the UGB / Mestrado / Matematica Aplicada / Mestre em Matemática Aplicada

This thesis encompasses two studies on the application of computational techniques, including deep learning and physics-based methods, in the exploration of protein structure and dynamics. In Chapter 1, I will introduce the background knowledge. Chapter 2 describes the development of a deep learning method for protein loop modeling. We introduce a fast and accurate method for protein loop structure modeling and refinement using deep learning. This method, which is both fast and accurate, integrates a protein language model, a graph neural network, and attention-based modules to predict all-atom protein loop structures from sequences. Its accuracy was validated on benchmark datasets CASP14 and CAMEO, showing performance comparable to or better than the state-of-the-art method, AlphaFold2. The model’s robustness against loop structures outside of the training set was confirmed by testing on datasets after removing high-identity templates and train- ing set homologs. Moreover, it demonstrated significantly lower computational costs compared to existing methods. Application of this method in real-world scenarios included predicting anti- body complementarity-determining regions (CDR) loop structures and refining loop structures in inexact side-chain environments. The method achieved sub-angstrom or near-angstrom accuracy for most CDR loops and notably enhanced the quality of many suboptimal loop predictions in in- exact environments, marking an advancement in protein loop structure prediction and its practical applications. Chapter 3 presents a collaborative study that employs nuclear magnetic resonance (NMR) experiments, molecular dynamics (MD), and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations to investigate protein conformational dynamics across varying temperatures. NMR chemical shifts provide a sensitive probe of protein structure and dynamics. Prediction of shifts, and therefore interpretation of shifts, particularly for the frequently measured amidic 15N sites, remains a tall challenge. We demonstrate that protein ¹⁵N chemical shift prediction from QM/MM predictions can be improved if conformational variation is included via MD sampling, focusing on the antibiotic target, E. coli Dihydrofolate reductase (DHFR). Variations of up to 25 ppm in predicted ¹⁵N chemical shifts are observed over the trajectory. For solution shifts, the average of fluctuations on the low picosecond timescale results in a superior prediction to a single optimal conformation. For low-temperature solid-state measurements, the histogram of predicted shifts for locally minimized snapshots with specific solvent arrangements sampled from the trajectory explains the heterogeneous linewidths; in other words, the conformations and associated solvent are ‘frozen out’ at low temperatures and result in inhomogeneously broadened NMR peaks. We identified conformational degrees of freedom that contribute to chemical shift variation. Backbone torsion angles show high amplitude fluctuations during the trajectory on the low picosecond timescale. For a number of residues, including I60, 𝝍 varies by up to 60o within a conformational basin during the MD simulations, despite the fact that I60 (and other sites studied) are in a secondary structure element and remain well folded during the trajectory. Fluctuations in 𝝍 appear to be compensated by other degrees of freedom in the protein, including 𝝓 of the succeeding residue, resulting in “rocking” of the amide plane with changes in hydrogen bonding interactions. Good agreement for both room-temperature and low-temperature NMR spectra provides strong support for the specific approach to conformational averaging of computed chemical shifts.

<p dir="ltr">The yeast zinc metalloprotease, Ste24, is involved in the maturation of the yeast mating pheromone <b>a</b>-factor by performing two distinct cleavages in the same precursor peptide substrate. Firstly, during the CaaX processing, Ste24 cleaves the three terminal residues of <b>a</b>-factor. CaaX processing is a well-studied process that involves the prenylation, proteolysis, and carboxyl-methylation of proteins ending with a cysteine (C), two aliphatic residues (aa), and one of several amino acids (X). The second cleavage step by Ste24 occurs after CaaX processing and involves an upstream cleavage N-terminal to the CaaX site. Another cleavage is performed by the enzyme Axl1 before the precursor peptide is transported from the cell to initiate mating processes. Inhibition of Ste24 typically results in ‘sterile’ cells which is how the term ‘Sterile 24’ was coined. In humans, defects in this metalloprotease or its substrate, Prelamin A, typically result in a range of progeroid disorders. Furthermore, the severity of these diseases has been directly linked to the catalytical activity of the enzyme. Treatments for these diseases are difficult to develop due to the limited knowledge available on the catalysis, substrate recognition, and functions of Ste24 and its homolog.</p><p dir="ltr">As such, these studies aim to define the substrate specificity of Ste24 and elucidate the binding site of Ste24. Identifying the substrate requirements of Ste24 has been an increasingly interesting topic due to the implication of Ste24 in a variety of unrelated functions. Previously, it has only been shown that yeast Ste24 is able to cleave the native substrate, the precursor of <b>a</b>-factor, and the substrate of its human homolog, prelamin A. This is an interesting finding because both substrates have dissimilar sequences at each cleavage site; so, it could be hypothesized that Ste24 may be able to recognize a wider range of sequences than expected. Further research has provided evidence that Ste24 is able to cleave both prenylated and non-prenylated substrates. It is also able to act as a translocon unclogger which may support its function in cleaving toxic islet amyloid polypeptides involved in cell failure in diabetes. Surprisingly, it was shown that this ‘unclogger ability’ was directly correlated to the activity level of Ste24, suggesting that the active site is directly involved in cleaving these peptides. With this information, it is clear that Ste24 has a broader substrate recognition ability than previously believed.</p><p dir="ltr">To elucidate the substrate specificity of Ste24, short peptide sequences containing varying CaaX sequences were developed and tested for C-terminal activity through a radioactive methyltransferase-coupled diffusion assay. Ste24 was able to recognize several sequences, however, a larger library is necessary to identify the specific requirements necessary for cleavage. Secondly, we tested the necessity of carboxylmethylation for the upstream N-terminal cleavage. The Distefano group designed three 33-mer analogs of <b>a</b>-factor, developed to mimic the C-terminally cleaved peptide. These peptides had either <b>a)</b> a methyl ester terminus representing the native substrate, <b>b)</b> a free carboxyl terminus representing the unmethylated precursor, and <b>c)</b> an amide terminus representing an unnatural end. All three peptides were tested using a FRET-based assay that allowed for the kinetic parameters of each peptide to be evaluated. We demonstrated that carboxylmethylation was not necessary for the upstream N-terminal cleavage; all three peptides presented similar kinetics. Finally, we interrogated the binding site of Ste24 through the use of a radioactive methyltransferase-coupled diffusion assay (C-terminal cleavage), a FRET-based assay (N-terminal cleavage), and photocrosslinking assays (binding). Together, these data presented a clearer image of residues necessary for the cleavage and binding of substrates within Ste24.</p>

Leung Ka-Kit. / Thesis submitted in: November 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 67-71). / Abstracts in English and Chinese. / Abstract / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background of protein structures --- p.1 / Chapter 1.2 --- Background of protein-protein interaction (PPI) --- p.4 / Chapter 1.2.1 --- Quaternary structure and protein complex --- p.4 / Chapter 1.2.2 --- Previous related work --- p.4 / Chapter 1.2.3 --- The kinetic and thermodynamic formalism --- p.6 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.10 / Chapter 2.1 --- Amino acid composition representative power modeling --- p.10 / Chapter 2.1.1 --- Propensity level modeling --- p.10 / Chapter 2.1.2 --- Polar atoms visualization --- p.17 / Chapter 2.2 --- Rigid structure representative power modeling --- p.17 / Chapter 2.3 --- Electrostatic potential modeling --- p.17 / Chapter 2.3.1 --- Charge residence --- p.17 / Chapter 2.3.2 --- Minimum Ribbon (MR) --- p.19 / Chapter 2.4 --- Examination of interface --- p.23 / Chapter 2.5 --- Identification procedures of a binding site --- p.24 / Chapter 2.6 --- System requirements --- p.24 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSIONS --- p.24 / Chapter 3.1 --- Polar atoms --- p.25 / Chapter 3.2 --- Minimum Ribbon (MR) --- p.27 / Chapter 3.3 --- "Charge complementarity, propensity level and rigid structure orientation" --- p.31 / Chapter 3.4 --- Identification of interacting site --- p.36 / Chapter CHAPTER 4. --- CONCLUSIONS --- p.64 / System requirements --- p.65 / Basic operation --- p.65 / Limitation --- p.66

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.

The goal of the following thesis research was to develop a systematic approach for the refinement of low-resolution protein models, as a part of the protein structure prediction procedure. Significant progress has been made in the field of protein structure prediction and the contemporary methods are able to assemble correct topology for a large fraction of protein domains. But such approximate models are often not detailed enough for some important applications, including studies of reaction mechanisms, functional annotation, drug design or virtual ligand screening. The development of a method that could bring those structures closer to the native is then of great importance. The minimal requirements for a potential that can refine protein structures is the existence of a correlation between the energy with native similarity and the scoring of the native structure as being lowest in energy. Extensive tests of the contemporary all-atom physics-based force fields were conducted to assess their applicability for refinement. The tests revealed flatness of such potentials and enabled the identification of the key problems in the current approaches. Guided by these results, the optimization of the AMBER (ff03) force field was performed that aimed at creating a funnel shape of the potential, with the native structure at the global minimum. Such shape should facilitate the conformational search during refinement and drive it towards the native conformation. Adjusting the relative weights of particular energy components, and adding an explicit hydrogen bond potential significantly improved the average correlation coefficient of the energy with native similarity (from 0.25 for the original ff03 potential to 0.65 for the optimized force field). The fraction of proteins for which the native structure had lowest energy increased from 0.22 to 0.90. The new, optimized potential was subsequently used to refine protein models of various native-similarity. The test employed 47 proteins and 100 decoy structures per protein. When the lowest energy structure from each trajectory was compared with the starting decoy, we observed structural improvement for 70% of the models on average. Such an unprecedented result of a systematic refinement is extremely promising in the context of high-resolution structure prediction.

Orientador: Mário Tyago Murakami / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T16:32:04Z (GMT). No. of bitstreams: 1 Martins_NadiaHelena_D.pdf: 33217097 bytes, checksum: 6cbd9ad5dcfddd06e2357b3c680cd3f6 (MD5) Previous issue date: 2011 / Resumo: A enzima purina nucleosídeo fosforilase hexamérica de Bacillus subtilis (BsPNP233) c uma nucleosídeo fosforilase do tipo 1 , responsável pela catalise reversível da reação de guebra de urn nucleosídeo em base nitrogenada e ribose-1-fosfato na via de salvação de purinas. Essa enzima possui interesses biomédicos e biotecnológicos devido ao uso na terapia gênica em canceres sólidos e na biossíntese de análogos de nucleosídeos...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The hexamcric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) is a nucleoside phosphorylase typc-1 involved in purine salvage pathway by the nucleoside phosphorolysis resulting in purine base and ribose-1-phosphatc. The interest about this enzyme involves gene therapy application in solid cancers treatment and nucleoside analogs biosynthesis...Note: The complete abstract is available with the full electronic document / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

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We have shown that nuclear matrix protein 4 (Nmp4) attenuates the response to intermittent parathyroid hormone (PTH) in healthy and ovariectomized (OVX) female mice using a global knockout of the Nmp4 gene. Additionally, these mice have increased bone marrow osteoprogenitors and CD8+ T-cells which support osteoblast differentiation. The animals were not protected from bone loss following OVX, but retained the hypersensitivity seen in the intact mice. Mesenchymal stem/progenitor cells (osteoprogenitors) demonstrated increased growth rate in culture and showed more robust differentiation into mineralizing bone cells. Chromosome precipitation followed by next generation sequencing and bioinformatics analysis characterized Nmp4 as a negative regulator of synthetic processes and suggested the IGF1/Akt and BMP2/Smad biochemical pathways which are likely targets for Nmp4 regulation. We have experimentally verified these pathways in immortalized bone marrow mesenchymal cells from wild type and Nmp4-KO mice. Disabling Nmp4 in estrogen replete or depleted mice confers an enhanced bone formation from intermittent parathyroid hormone.

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