The work presented in this thesis focusses primarily on the determination of protein structure at atomic resolution, with NMR spectroscopy as the principle investigative tool. The thesis is divided into four parts. Part I consists of Chapter 1 which provides an introduction to protein structure, folding and NMR spectroscopy. Part II, consisting of Chapters 2 and 3, describes the effects of aromatic interactions on nucleating structure in disordered regions of proteins, using variants of apo-cytochrome b5 as a model system. Part III consists of Chapter 4, which describes structural effects of introducing cross-strand disulfide bonds using variants of Thioredoxin. Part IV of this thesis consists of the Appendices A, B and C. Appendix A describes the purification and characterization of ilvM, the regulatory subunit of the E.coli enzyme AHAS II. Appendices B and C contain chemical shift information corresponding to Chapter 3 and Chapter 4 respectively. Part I : Introduction to protein structure, folding and solution structure studies Chapter 1 first gives a brief overview of protein structure followed by an introduction to protein folding, focussing on the forces involved in determining the final three-dimensional shape of the protein as well as the experimental and computational techniques involved in studying or predicting the fold of a given protein. The second section of this chapter details the methodology followed to obtain solution structures of proteins using NMR spectroscopy. Part II : Engineering aromatic interactions to nucleate folding in intrinsically disordered regions of proteins Chapter 2 describes site-specific mutagenesis, recombinant over-expression, purifica-tion and preliminary biophysical characterization of two aromatic mutants of the molten globule apo-cytochrome b5 (apocytb5) : H43F H67F cytochrome b5 (FFcytb5) and H43W H67F cytochrome b5 (WFcytb5). Analysis of the structure of wild-type apo - cytochrome b5 was done to introduce surface mutations and avoid perturbation of the interior pack-ing of the protein. The bacterial host E.coli BL21(DE3) was used for recombinant over-expression, and both mutant proteins were purified by anion-exchange chromatography followed by size-exclusion chromatography. Biophysical studies show a decrease in the hydrodynamic radii and surface hydropho-bicity of FFcytb5 and WFcytb5 compared to wt -apo cytb5. An increase in protein stability was also seen from the wt apocytb5 to WFcytb5 and FFcytb5 in the presence of the chemical denaturant Urea. Proton 1D NMR spectra exhibited sharp lines and good spectral dispersion in the amide region, indicating that both mutant proteins are well folded. In addition, conservation of two distinctive up field and downfield shifted resonances present in apocytb5 indicated that structural changes upon mutation accrued on the upon the scaffold of apocytb5. Chapter 3 describes solution structure studies to determine secondary and tertiary structure of FFcytb5 and WFcytb5. Structural studies were carried out using homonu-clear and heteronuclear NMR methods, for which isotopically enriched 15N- and 13C, 15N samples were prepared for each protein. Additionally a 2H, 13C, 15N ILV methyl labeled sample was prepared for FFcytb5 to obtain unambiguous NOE correlation data. The hydrogen bond network for WFcytb5 was determined using hydrogen/deuterium exchange data. The restraints required to define the orientations and interactions of the aromatic groups were obtained from 15N-edited NOESY HSQC, 13C -edited NOESY HSQC and 2D 1H - 1H NOE spectra. These correlations were crucial in determining the aromatic interactions present within each protein. The structure of FFcytb5 was calculated using 1163 NOE distance restraints, 179 φ and ψ dihedral angle restraints, along with 40 hydrogen bond restraints. Similarly the structure of WFcytb5 was calculated using 1282 NOE distance restraints, 177 φ and ψ dihedral angle restraints and 40 hydrogen bond restraints. The ensemble of structures obtained for FFcytb5 showed a root mean square deviation of 1.01±0.21 Å . The ensemble of structures obtained for WFcytb5 showed a root mean square deviation of 0.58±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were found to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The final tertiary structure of both FFcytb5 and WFcytb5 consisted of a mixed four strand β -sheet with a four helix bundle resting on top and were seen to align well, with an RMSD of 0.6 Å. A comparison of the solution structures of apocytb5 with FFcytb5 and WFcytb5 convincingly showed the nucleation secondary and tertiary structure well beyond the site of mutation. The presence of aromatic trimers, non-canonical in context of the wt apoc-ytb5, was confirmed upon analysis of the structures of FFcytb5 and WFcytb5, with NOE correlations assigned to verify these interactions. The reduction in the hydrodynamic radii of FFcytb5 and WFcytb5 in relation to apocytb5 was also verified from tsuperscript15N-NMR relaxometry studies. The nucleation of long-range structure using aromatic interactions has been demonstrated in proteins for the first time, and can in principle be used to incorporate aromatic residues and interactions in protein design. Structural data, chemical shift data and restraints lists used for structure calculation of WFcytb5 and FFcytb5 were deposited with the PDB (accession numbers 5XE4 and 5XEE) and BMRB(accession numbers 36070, 36071) respectively1. Part III : Structural consequences of introducing disulfide bonds into β - sheets Chapter 3 describes the solution structure studies on two mutants of E.coli Thiore-doxin which were designed to incorporate a disulfide bond between two anti-parallel β-strands at the edge of the β-sheet. One mutant was designed with a disulfide bond at the hydrogen bonding position (HB, 78c90cTrx) and the other with the disulfide bond at the non-hydrogen bonding position (NHB, 77c91cTrx). Here we study the structural changes that accompany the introduction of a cross-strand disulfide and whether such structural changes could be correlated with the previously seen thermodynamic and catalytic changes. Solution structure studies were conducted using a suite of multidimensional heteronu-clear NMR experiments, for which isotopically enriched 15N and 13C, 15N labelled samples were used. The solution structure for 77c91cTrx was calculated using 1190 NOE distance restraints, 199 φ and ψ dihedral angle restraints and 48 hydrogen bond restraints. The solution structure for 78c90cTrx was calculated using 1123 NOE distance restraints, 197 φ and ψ dihedral angle restraints and 50 hydrogen bond restraints. The ensemble of structures for 77c91cTrx showed an RMSD of 0.78± 0.13 Å while the RMSD for the ensemble of structures of 78c90cTrx was seen to be 0.76±0.09 Å . In both cases, ≈ 80% of backbone dihedral angles were seen to be in the allowed regions and ≈ 20% in the additionally allowed regions of the Ramachandran map. The tertiary structures of both proteins were seen to have a 5-strand mixed β-sheet and 4 helices surrounding it. . A comparison of the solution structures of mutant and wt -Trx showed significant changes in secondary and tertiary structure. For example, an α helix was reduced from 3 turns to a single turn, and of the β-strands containing the mutation was elongated by 3 residues. A ≈ 50% loss of hydrogen bonds, primarily from the β -sheet, was seen for both mutants. The secondary and tertiary structure for both 77c91cTrx and 78c90cTrx was seen to be near identical, despite the greater strain of the disulfide bond at the hydrogen bonding position. In addition to this, the Ile75-Pro76 peptide bond is now seen to be in the trans conformation in 78c90cTrx, while in wt -Trx the Ile75-Pro76 peptide bond is in the cis conformation. This cis peptide bond is known to play a role in both folding and catalysis, and the solution structures were analyzed in the context of observed changes in folding and catalysis. The study shows that introducing disulfide bonds even at the edge of β sheets have long-range structural effects, and the structural effects cannot be directly correlated with the changes in stability. Part III: Appendix Appendix A describes the expression, purification and preliminary characterization of ilvM, the regulatory subunit of E.coliAHAS II, one of three enzyme isomers that catal-yse the first step in the synthesis of all branched chain amino acids. AHAS II is known to be insensitive to feedback regulation, but our studies showed that the presence of Ile, Leu and Val causes structural changes and increases the stability of ilvM. However we were not able to purify ilvM in sufficient quantities to proceed with solution structure studies. Appendices B and C contain chemical shift information for the structural studies carried out on FFcytb5, WFcytb5, 77c91cTrx and 78c90cTrx.

abstract: Spider dragline silk is an outstanding biopolymer with a strength that exceeds steel by weight and a toughness greater than high-performance fibers like Kevlar. For this reason, structural and dynamic studies on the spider silk are of great importance for developing future biomaterials. The spider dragline silk comprises two silk proteins, Major ampullate Spidroin 1 and 2 (MaSp1 and 2), which are synthesized and stored in the major ampullate (MA) gland of spiders. The initial state of the silk proteins within Black Widow MA glands was probed with solution-state NMR spectroscopy. The conformation dependent chemical shifts information indicates that the silk proteins are unstructured and in random coil conformation. 15N relaxation parameters, T1, T2 and 15N-{1H} steady-state NOE were measured to probe the backbone dynamics for MA silk proteins. These measurements indicate fast sub-nanosecond timescale backbone dynamics for the repetitive core of spider MA proteins indicating that the silk proteins are unfolded, highly flexible random coils in the MA gland. The translational diffusion coefficients of the spider silk proteins within the MA gland were measured using 1H diffusion NMR at 1H sites from different amino acids. A phenomenon was observed where the measured diffusion coefficients decrease with an increase in the diffusion delay used. The mean displacement along the external magnetic field was found to be 0.35 μm and independent of the diffusion delay. The results indicate that the diffusion of silk protein was restricted due to intermolecular cross-linking with only segmental diffusion observable. To understand how a spider converts the unfolded protein spinning dope into a highly structured and oriented in the super fiber,the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. The in vitro spider silk assembly kinetics were monitored as a function of pH with a 13C solid-state Magic Angle Spinning (MAS) NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation and the percentage of β-sheet structure in the grown fibers depend on pH. The secondary structure of the major ampullate silk from Peucetia viridians (Green Lynx) spiders was characterized by X-ray diffraction (XRD) and solid-state NMR spectroscopy. From XRD measurement, β-sheet nano-crystallites were observed that are highly oriented along the fiber axis with an orientational order of 0.980. Compare to the crystalline region, the amorphous region was found to be partially oriented with an orientational order of 0.887. Further, two dimensional 13C-13C through-space and through-bond solid-state NMR experiments provide structural analysis for the repetitive amino acid motifs in the silk proteins. The nano-crystallites are mainly alanine-rich β-sheet structures. The total percentage of crystalline region is determined to be 40.0±1.2 %. 18±1 % of alanine, 60±2 % glycine and 54±2 % serine are determined to be incorporated into helical conformations while 82±1 % of alanine, 40±3 % glycine and 46±2 % serine are in the β-sheet conformation. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

A contração muscular esquelética é regulada pelo complexo troponina/tropomiosina de maneira dependente de Ca2+. O complexo troponina consiste de três subunidades: a troponina C (TnC), a troponina I (TnI) e a troponina T (TnT). A troponina C é a subunidade que liga Ca2+, a TnI é a subunidade inibitória e a TnT liga-se fortemente à tropomiosina. A TnI e a TnT são altamente insolúveis a baixas forças iônicas, a não ser que estejam complexadas com a TnC. O complexo troponina pode ser reconstituído \"in vitro\" a partir das subunidades isoladas simplesmente misturando-se as subunidades em razões equimolares em uréia, que depois é removida através de diálise. Na primeira parte deste trabalho um vetor para a co-expressão da TnC, TnI e TnT em E.coli foi construído. Utilizando este vetor nós produzimos um complexo troponina funcional montado no citoplasma de E.coli. A presença da TnT é requerida para regulação dependente de Ca2+ da contração muscular esquelética. O papel da TnT em conferir sensibilidade ao Ca2+ à atividade ATPásica da acto-miosina foi analisado. Mutantes de deleção da TnT foram construídos através de mutação sítio-dirigida e expressos em E.coli. Complexos troponina contendo os mutantes de TnT e/ou mutantes de TnI foram reconstituídos e analisados em ensaios de ligação ao filamento fino e ensaios de atividade ATPásica. Baseado nestes resultados a TnT foi subdividida em três domínios: o domínio ativatório (aminoácidos 157-216), o domínio inibitório (aminoácidos 157-216) e o domínio de ancoragem do dímero TnC/TnI (aminoácidos 216-263). Nós demonstramos que o dímero TnC/TnI está ancorado ao filamento fino através da interação entre a região amino-terminal da TnI e da região carbóxi-terminal da TnT (aminoácidos 216-263). Um modelo para o papel da TnT na regulação da contração muscular dependente de Ca2+ é proposto. / The contraction of skeletal muscle is regulated by troponin and tropomyosin in a Ca2+ dependent manner. The troponin complex consists of three subunits: troponin C (TnC), troponin I (TnI) and troponin T (TnT). Troponin C is the Ca2+ binding subunit, TnI is the inhibitory subunit and TnT binds tightly to tropomyosin. TnI and TnT are highly insoluble proteins at low ionic strengths, unless they are complexed with TnC. The troponin complex can be reconstituted \"in vitro\" from the isolated subunits simply by mixing the subunits at equimolar ratios in urea, which is then removed by dialysis. In the first part of this work a vector for the co-expression of TnC, TnI and TnT in E.coli was constructed. Using this vector we were able to produce a functional troponin complex assembled \"in vivo\" in the E.coli cytoplasm The presence of TnT is required for the Ca2+ dependente regulation of the skeletal muscle contraction. The role of TnT in conferring full Ca2+ sensitivity to the ATPase activity of acto-myosin was analyzed. Deletion mutants of TnT were constructed by site-directed mutagenesis and expressed in E.coli. Troponin complexes containing the TnT deletion mutants and/or TnI deletion mutants, were reconstituted and analyzed in thin filament binding assays and in ATPase activity assays. Based on these studies, TnT was subdivided into three domains: the activation domain (comprised of aminoacids 1-157), the inhibitory domain (comprised of amino acids 157-216) and the TnC/TnI dimer anchoring domain (aminoacids 216-263). We demonstrated that the TnC/TnI is anchored to the thin filament through interaction between the amino-terminal domain of TnI and the region comprised of aminoacids 216-263 of TnT. A model for the role of TnT in the Ca2+ dependent regulation of muscle contraction is proposed.

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Proteínas desempenham uma grande variedade de funções biológicas. O conhecimento da estrutura tridimensional proteica pode ajudar no entendimento da função desempenhada. De acordo com a hipótese de Anfisen, a estrutura terciária nativa de uma proteína pode ser determinada a partir da informação contida na sequência primária, o que permitiria que métodos computacionais poderiam ser usados para predizer estruturas terciárias quando a primária estiver disponível. No entanto, ainda não existe uma ferramenta computacional capaz de predizer a estrutura tridimensional para uma grande variedade de proteínas. Desse modo, o problema de Predição de Estruturas de Proteínas (PEP) permanece como um desafio para a Biologia Molecular. A conformação nativa de uma proteína é frequentemente a configuração termodinamicamente mais estável, ou seja, que possui menor energia livre. Assim, PEP pode ser vista como um problema de otimização, onde a estrutura com menor energia livre deve ser encontrada dentre todas as possíveis. Entretanto, este é um problema NP-completo, no qual métodos tradicionais de otimização, em geral, não apresentam um bom desempenho. Algoritmos Genéticos (AGs), devido às suas características, são interessantes para essa classe de problemas. O principal objetivo desse trabalho é verificar se a adição de informação pode ser útil aos AGs aplicados em PEP, valendo-se dede modelos moleculares simplificados. Cada indivíduo do AG representa uma solução que, neste caso, é uma possível conformação que será avaliada por um campo de força. Dessa forma, o indivíduo é codificado por um conjunto de ângulos de torção de cada aminoácido. Para auxiliar no processo de busca, bases de dados compostas de ângulos determinados por cristalografia e RNM são utilizadas. Com o objetivo de guiar o processo de busca e manter a diversidade nos AGs, duas estratégias são aqui testadas: Imigrantes Aleatórios e Imigrantes por Similaridade. A última delas foi criada baseando-se na similaridade da sequência primária. Além disso, é investigado neste trabalho o uso de um campo de força coarse grained, que utiliza os átomos de carbono- para representar a cadeia proteica, para avaliar os indivíduos do AG. / Proteins exhibit an enormous variety of biology functions. The knowledge of tertiary structures can help the understanding of the proteins function. According to Anfisen, the native tertiary structure of a protein can be determined by its primary structure information, what could allow that computational methods could be used to predict the tertiary structure when the primary structure is available. However, there is still not a computational tool to solve the structure prediction problem for a large range of proteins. In this way, Protein Structure Prediction (PSP) has been a challenge to Molecular Biology. The conformation of native protein is usually the thermodynamically most stable configuration, i.e., the one having the lowest free energy. Hence, PSP can be viewed as a problem of optimization, where the structure with the lowest free energy should be found among all possible structures. However, this is an NP-problem, where traditional optimization methods, in general, do not have good performance. Genetic algorithms (GAs), due to their characteristics, are interesting for this class of problems. In recent years, there is a growing interest in using GAs for the protein structure prediction problem. The main objective of this work is to verify the addition of useful information to GAs employed in PSP. Each individual of the GA represents a solution for the optimization problem which is, in this case, a possible conformation that will be evaluated by a force field function. Thus, an individual is encoded by a set of torsion angles of each amino acid. In order to reduce the search space, a database composed of angles, determined by crystallography and NMR, is used. With the aim to guide the final search process and maintain diversity in GAs, two strategies were employed here: Random Immigrants and Similarity-based Immigrants. The last strategy was based on similarity of primary amino acid sequence. Furthermore, in this work, a coarse-grained force field, which uses -carbon to represent the protein backbone was employed to evaluate the individuals of GA.

Due to the inherent problems with their structural elucidation in the laboratory, the computational prediction of membrane protein structure is an essential step toward understanding the function of these leading targets for drug discovery. In this work, the development of a genetic algorithm technique is described that is able to generate predictive 3D structures of membrane proteins in an ab initio fashion that possess high stability and similarity to the native structure. This is accomplished through optimisation of the distances between TM regions and the end-on rotation of each TM helix. The starting point for the genetic algorithm is from the model of general TM region arrangement predicted using the TMRelate program. From these approximate starting coordinates, the TMBuilder program is used to generate the helical backbone 3D coordinates. The amino acid side chains are constructed using the MaxSprout algorithm. The genetic algorithm is designed to represent a TM protein structure by encoding each alpha carbon atom starting position, the starting atom of the initial residue of each helix, and operates by manipulating these starting positions. To evaluate each predicted structure, the SwissPDBViewer software (incorporating the GROMOS force field software) is employed to calculate the free potential energy. For the first time, a GA has been successfully applied to the problem of predicting membrane protein structure. Comparison between newly predicted structures (tests) and the native structure (control) indicate that the developed GA approach represents an efficient and fast method for refinement of predicted TM protein structures. Further enhancement of the performance of the GA allows the TMGA system to generate predictive structures with comparable energetic stability and reasonable structural similarity to the native structure.

Evolutionary algorithms (EAs) have been shown to be competent at solving complex, multimodal optimisation problems in applications where the search space is large and badly understood. EAs are therefore among the most promising classes of algorithms for solving the Protein Structure Prediction Problem (PSPP). The PSPP is how to derive the 3D-structure of a protein given only its sequence of amino acids. This dissertation defines, evaluates and shows limitations of simplified models for solving the PSPP. These simplified models are off-lattice extensions to the lattice HP model which has been proposed and is claimed to possess some of the properties of real protein folding such as the formation of a hydrophobic core. Lattice models usually model a protein at the amino acid level of detail, use simple energy calculations and are used mainly for search algorithm development. Off-lattice models usually model the protein at the atomic level of detail, use more complex energy calculations and may be used for comparison with real proteins. The idea is to combine the fast energy calculations of lattice models with the increased spatial possibilities of an off-lattice environment allowing for comparison with real protein structures. A hypothesis is presented which claims that a simplified off-lattice model which considers other amino acid properties apart from hydrophobicity will yield simulated structures with lower Root Mean Square Deviation (RMSD) to the native fold than a model only considering hydrophobicity. The hypothesis holds for four of five tested short proteins with a maximum of 46 residues. Best average RMSD for any model tested is above 6Å, i.e. too high for useful structure prediction and excludes significant resemblance between native and simulated structure. Hence, the tested models do not contain the necessary biological information to capture the complex interactions of real protein folding. It is also shown that the EA itself is competent and can produce near-native structures if given a suitable evaluation function. Hence, EAs are useful for eventually solving the PSPP.

A novel approach is proposed for modeling of structurally variable regions in proteins. In this approach, a prerequisite sequence-structure alignment is examined for regions where the target sequence is not covered by the structural template. These regions, extended with a number of residues from adjacent stem regions, are submitted to fold recognition. The alignments produced by fold recognition are integrated into the initial alignment to create a multiple alignment where gaps in the main structural template are covered by local structural templates. This multiple alignment is used to create a protein model by existing protein modeling techniques. Several alternative parameters are evaluated using a set of ten proteins. One set of parameters is selected and evaluated using another set of 31 proteins. The most promising result is for loop regions not located at the C- or N-terminal of a protein, where the method produces an average RMSD 12% lower than the loop modeling provided with the program MODELLER. This improvement is shown to be statistically significant.

An experimental study on randomly generated protein sequences can provide important insights into the origin and mechanism of secondary structure formation and protein folding. In this study we bring biophysical characterization of five protein sequences selected from the in silico generated library of random chains. The sequences were selected on the basis of bioinformatic analysis in order to find the candidates with the maximum potential to possess secondary structure. This study shows that the random polypeptide sequences form stable secondary structures and in some show the signs of tertiary structure, such as hydrophobic core formation and distinctive oligomerization pattern. While the work presented in this thesis is work in progress on a larger study, the data already demonstrate that unevolved protein sequence space provides a lot of potential for secondary and tertiary structure formation that awaits its characterization. Powered by TCPDF (www.tcpdf.org)