The atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 has recently been reported[1]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints measured on the ‘invisible’ intermediate state using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the data-base structure determination program CS-Rosetta. This thesis focuses on the cross-validation of the structure so produced. We present here the solution NMR structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by protein mutagenesis. The ensemble of structures generated of the mimic are within 2Å of the relaxation dispersion/CS-Rosetta structures of the intermediate, with the non-native interactions in the intermediate also observed in the mimic. The results presented in this thesis strongly confirm the structure of the FF domain folding intermediate, in particular, and validate the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

The atomic resolution structure of a low populated and transiently formed on-pathway folding intermediate of the FF domain from human HYPA/FBP11 has recently been reported[1]. The structure was determined on the basis of backbone chemical shift and bond vector orientation restraints measured on the ‘invisible’ intermediate state using relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy that were subsequently input into the data-base structure determination program CS-Rosetta. This thesis focuses on the cross-validation of the structure so produced. We present here the solution NMR structure of a mimic of the folding intermediate that is highly populated in solution, obtained from the wild-type domain by protein mutagenesis. The ensemble of structures generated of the mimic are within 2Å of the relaxation dispersion/CS-Rosetta structures of the intermediate, with the non-native interactions in the intermediate also observed in the mimic. The results presented in this thesis strongly confirm the structure of the FF domain folding intermediate, in particular, and validate the use of relaxation dispersion derived restraints in structural studies of invisible excited states, in general.

Anion exchanger 1 (AE1) is a red cell membrane glycoprotein that associates with cytoskeletal protein 4.2 in a complex bridging the cell membrane to the cytoskeleton. Disruption of this linkage results in unstable erythrocytes and hereditary spherocytosis (HS). Three HS mutations (E40K, G130R and P327R) in the cytoplasmic domain of AE1 (cdAE1) result in a decreased level of protein 4.2 in the red cell yet maintain normal amounts of AE1. Biophysical analyses showed the HS mutations had little effect on the structure and conformational stability of the isolated domain. However, the conformation of the cytoplasmic domain of the kidney anion exchanger, lacking the first 65 amino acids including a central -strand, was thermally destabilized relative to cdAE1 and had a more open structure. In transfected human embryonic kidney (HEK)-293 cells the HS mutants had similar expression levels as wild-type AE1, and protein 4.2 expression level was not dependent on the presence of AE1. Protein 4.2 localized to the plasma membrane with wild-type AE1, the HS mutants of AE1, the membrane domain of AE1 and kidney AE1, and to the ER with Southeast Asian ovalocytosis AE1. A fatty acylation mutant of protein 4.2, G2A/C173A, could not localize to the plasma membrane in the absence of AE1. Subcellular fractionation showed wild-type and G2A/C173A protein 4.2 were mostly associated with the cytoskeleton. Co-immunoprecipitation and Ni-NTA pull-down assays revealed impaired binding of protein 4.2 to HS mutants compared to AE1, while the membrane domain of AE1 was unable to bind protein 4.2. These studies show that HS mutations in cdAE1 cause impaired binding of protein 4.2, without causing gross structural changes in the domain. The mutations change the binding surface on cdAE1 by the introduction of positive charges into an otherwise acidic domain. This binding impairment may render protein 4.2 more susceptible to degradation or loss during red cell development.

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.

The structural determination of interacting proteins, both as individual proteins and in their complex, complemented by thermodynamical studies are vital in order to gain in-depth insights of the phenomena leading to the highly selective protein-protein interactions characteristic of numerous life processes. This thesis describes an investigation of the structural and thermodynamical basis for molecular recognition in two different protein-protein complexes, formed between so-called affibody proteins and their respective targets. Affibody proteins are a class of engineered binding proteins, which can be functionally selected for binding to a given target protein from large collections (libraries) constructed via combinatorial engineering of 13 surface-located positions of the 58-residue three-helix bundle Z domain derived from Staphylococcal protein (SPA). In a first study, an affibody:target protein pair consisting of the ZSPA-1 affibody and the parental Z domain, with a dissociation constant (Kd) of approximately 1 µM, was investigated. ZSPA-1 was in its free state shown to display molten globule-like characteristics. The enthalpy change on binding between Z and ZSPA-1 as measured by isothermal titration calorimetry, was found to be a non-linear function of temperature. This nonlinearity was found to be due to the temperature dependent folded-unfolded equilibrium of ZSPA-1 upon binding to the Z domain and, the energetics of the unfolding equilibrium of the molten globule state of ZSPA-1 could be separated from the binding thermodynamics. Further dissection of the binding entropy revealed that a significant reduction in conformational entropy resulting from the stabilization of the molten globule state of ZSPA-1 upon complex formation could be a major reason for the moderate binding affinity. A second studied affibody:target complex (Kd ~ 0.1 µM) consisted of the ZTaq affibody protein originally selected for binding to Taq DNA polymerase and the anti-ZTaq affibody protein, selected for selective binding to the ZTaq affibody protein, thus constituting an "anti-idiotypic" affinity protein pair. The structure of the ZTaq:anti-ZTaq affibody complex as well as the free state structures of ZTaq and anti-ZTaq were determined using NMR spectroscopy. Both ZTaq and anti-ZTaq are well defined three helix bundles in their free state and do not display the same molten globule-like behaviour of ZSPA-1. The interaction surface was found to involve all of the varied positions in helices 1 and 2 of the anti-ZTaq, the majority of the corresponding side chains in ZTaq, and also several non-mutated residues. The total buried surface area was determined to about 1670 Å2 which is well inside the range of what is typical for many protein-protein complexes, including antibody:antigen complexes. Structural rearrangements, primarily at the side chain level, were observed to take place upon binding. There are similarities between the ZTaq:anti-ZTaq and the Z:ZSPA-1 structure, for instance, the binding interface area in both complexes has a large fraction of non-polar content, the buried surface area is of similar size, and certain residues have the same positioning. However, the relative orientation between the subunits in ZTaq:anti-ZTaq is markedly different from that observed in Z:ZSPA-1. The thermodynamics of ZTaq:anti-ZTaq association were investigated by isothermal titration calorimetry. A dissection of the entropic contributions showed that a large and favourable desolvation entropy of non-polar surface is associated with the binding reaction which is in good agreement with hydrophobic nature of the binding interface, but as in the case for the Z:ZSPA-1 complex a significant loss in conformational entropy opposes complex formation. A comparison with complexes involving affibody proteins or SPA domains suggests that affibody proteins inherit intrinsic binding properties from the original SPA surface. The structural and biophysical data suggest that although extensive mutations are carried out in the Z domain to obtain affibody proteins, this does not necessarily affect the structural integrity or lead to a significant destabilization. / QC 20110118

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 134-146). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

A demanda criada pelos estudos biológicos resultou para predição da estrutura terciária de proteínas ser uma alternativa, uma vez que menos de 1% das sequências conhecidas possuem sua estrutura terciária determinada experimentalmente. As predições Ab initio foca nas funções baseadas da física, a qual se trata apenas das informações providas pela sequência primária. Por consequência, um espaço de busca com muitos mínimos locais ótimos deve ser pesquisado. Este cenário complexo evidencia uma carência de algoritmos eficientes para este espaço, tornando-se assim o principal obstáculo para este tipo de predição. A optimização Multi-Objetiva, principalmente os Algoritmos Evolutivos, vem sendo aplicados na predição da estrutura terciária já que na mesma se envolve um compromisso entre os objetivos. Este trabalho apresenta o framework ProtPred-PEO-GROMACS, ou simplesmente 3PG, que não somente faz predições com a mesma acurácia encontrada na literatura, mas também, permite investigar a predição por meio da manipulação de combinações de objetivos, tanto no aspecto energético quanto no estrutural. Além disso, o 3PG facilita a implementação de novas opções, métodos de análises e também novos algoritmos evolutivos. A fim de salientar a capacidade do 3PG, foi então discorrida uma comparação entre os algoritmos NSGA-II e SPEA2 aplicados na predição Ab initio da estrutura terciária de proteínas em seis combinações de objetivos. Ademais, o uso da técnica de refinamento por Dinâmica Molecular é avaliado. Os resultados foram adequados quando comparado com outras técnicas de predições: Algoritmos Evolutivo Multi-Objetivo, Replica Exchange Molecular Dynamics, PEP-FOLD e Folding@Home. / The demand created by biological studies resulted the structure prediction as an alternative, since less than 1% of the known protein primary sequences have their 3D structure experimentally determined. Ab initio predictions focus on physics-based functions, which regard only information about the primary sequence. As a consequence, a search space with several local optima must be sampled, leading to insucient sampling of this space, which is the main hindrance towards better predictions. Multi-Objective Optimization approaches, particularly the Evolutionary Algorithms, have been applied in protein structure prediction as it involves a compromise among conicting objectives. In this paper we present the ProtPred-PEO-GROMACS framework, or 3PG, which can not only make protein structure predictions with the same accuracy standards as those found in the literature, but also allows the study of protein structures by handling several energetic and structural objective combinations. Moreover, the 3PG framework facilitates the fast implementation of new objective options, method analysis and even new evolutionary algorithms. In this study, we perform a comparison between the NSGA-II and SPEA2 algorithms applied on six dierent combinations of objectives to the protein structure. Besides, the use of Molecular Dynamics simulations as a renement technique is assessed. The results were suitable when comparated with other prediction methodologies, such as: Multi-Objective Evolutionary Algorithms, Replica Exchange Molecular Dynamics, PEP-FOLD and Folding@Home.

Orientador: Prof. Dr. Antônio Sérgio Kimus Braz / Dissertação (mestrado) - Universidade Federal do ABC. Programa de Pós-Graduação em Biossistemas, 2015. / O NGF (fator de crescimento neural) é uma proteína endógena secretada no sistema nervoso central e periférico, desempenhando diversos papéis, tais como neuroproteção, desenvolvimento e diferenciação neuronal, crescimento de neuritos e plasticidade sináptica. Sintetizado no neocórtex e no hipocampo, o NGF atua em populações de neurônios colinérgicos, sensoriais e simpáticos através da interação com receptores TrkA e p75NTR. Está relacionado com neuropatologias como doença de Alzheimer, hiperalgesia e com as neuropatologias autonômicas e sensoriais hereditárias tipos IV e V, onde foram identificas as mutações S187N e R221W, respectivamente. Apesar do vasto conhecimento da ação das vias relacionadas ao NGF e seus receptores, os mecanismos moleculares de interação, bem como da ativação das sinalizações internas dos receptores, ainda não estão totalmente esclarecidos. Através da abordagem de análise de modos normais, foram investigadas as alterações estruturais dos mutantes em relação ao tipo selvagem de NGF sob a ótica de estrutura de proteínas. Diferenças significativas na correlação de movimentos globais, flexibilidade do complexo, interações moleculares e na liberdade conformacional em ambas as mutações foram encontradas. Foram observadas variações estruturais e limitações conformacionais nos receptores como reflexo da ação dos mutantes de NGF, indicando que, mesmo após a interação, neurotrofina e receptor formam um complexo ativo. Desse modo, as mutações no NGF reduzem a capacidade de formação do complexo ativado através de alterações estruturais e conformacionais, causando prejuízo às funções biológicas derivadas da interação. / NGF (nerve growth factor) is an endogenous protein secreted in the central and peripheral nervous system, playing different roles, such as neuroprotection, development and neuronal differentiation, neurite outgrowth and synaptic plasticity. Synthesized in the neocortex and hippocampus, NGF acts on populations of cholinergic, sensory and sympathetic neurons through interaction with TrkA and p75NTR receptors. It is related neuropathologies such as Alzheimer's disease, hyperalgesia and hereditary sensory and autonomic neuropathologies types IV and V, which were identified the mutations S187N and R221W, respectively. Despite the vast knowledge of the action of pathways related to NGF and its receptors, the molecular mechanisms of interaction as well as the activation of the internal signalling pathways of the receptors are still not fully understood. Through the normal modes analysis approach, the structural changes of the mutants compared to wild type NGF were investigated from the perspective of protein structure. Significant changes in global movements correlation, complex flexibility, molecular interactions and conformational freedom were found for both mutations. Structural changes and conformational limitations in the receptors were observed, as reflecting the action of NGF mutants, indicating that even after the interaction neurotrophin and receptor form an active complex. Thus, mutations in the NGF reduce the ability of activated complex formation by structural and conformational changes, causing impairment to biological functions derived from the interaction.

A troponina C é o componente do complexo heterotrimérico troponina ao qual o Ca2+ se associa. Essa associação torna a contração muscular um processo regulado por Ca2+. Pearlstone et al. (Biochemistry 31, 6545, (1992)) utilizaram o cDNA da troponina C de músculo esquelético de galinha (sTnC) para construir um mutante onde a fenilalanina da posição 29 foi substituída por triptofano (mutante F29W). Esse mutante permitiu que a ligação de Ca2+ aos sítios regulatórios amino terminais fosse acompanhada através de técnicas fluorescentes. Entretanto, algumas propriedades da sTnC foram alteradas pela mutação (Li et al. (1995) Biochemistry 34, 8330). No presente estudo, ensaios de ligação direta de Ca2+ bem como titulações de Ca2+ seguidas por fluorescência foram usadas para melhor se entenderem os efeitos da mutação Phe→Trp na posição 29 bem como a influência de certos aminoácidos componentes do sítio de ligação de Ca2+ nas propriedades do domínio regulatório. Dois novos mutantes foram construídos nos quais os análogos do triptofano 5-hidroxitriptofano ou 7 -azatriptofano foram introduzidos na posição 29 (resultando nos mutantes F29HW e F29ZW, respectivamente). Os dados mostraram que, quando comparada com a sTnC, a afinidade por Ca2+ dos sítios amino terminais foi elevada na F29W em aproximadamente seis vezes, três vezes na F29ZW e levemente diminuída na F29HW . A curva de fluorescência associada à ligação de Ca2+ à F29ZW mostrou-se bimodal, com cada fase podendo ser relacionada à ligação de Ca2+ a cada um dos sítios regulatórios. Esta é a primeira descrição através de técnicas de fluorescência da ligação sequencial de Ca2+ aos sítios amino terminais. Para investigar a influência de cada um dos sítios amino terminais nas propriedades de ligação ao Ca2+ ou propriedades fluorescentes da sTnC, F29W, F29HW e F29ZW , construímos mutantes duplos e triplos através da substituição do aspartato da posição 30 ou 66 (ou ambos) por alanina. Essas mutações afetam respectivamente a capacidade de ligação ao Ca2+ dos sítios I e II. Os dados mostraram que: 1) nas concentrações de Ca2+ analisadas, a mutação Asp→Ala na posição 30 impede somente a ligação de Ca2+ ao sítio I, enquanto a mutação Asp → Ala na posição 66 impede a ligação de Ca2+ aos sítios I e II, e 2) a mutação Asp → Ala na posição 30 torna silenciosa a substituição da fenilalanina da posição 29 por Trp, 5-hidroxitriptofano ou 7-azatriptofano. Concluímos que o sítio I é essencialmente inativo sem a ligação prévia de Ca2+ ao sítio II e que a posição 29 influencia a afinidade ao Ca2+ do sítio I \"ajustado\". / Troponin C is the Ca2+ binding component of heterotrimeric troponin. It makes skeletal muscle contraction a Ca2+ regulated process. We have previously used the cDNA of chicken skeletal TnC (sTnC) to construct a mutant where phenylalanine at position 29 was replaced by tryptophan (F29W mutant) [Pearlstone et al. (1992) Biochemistry 31, 6545]. This mutant allowed calcium binding to the regulatory amino terminal sites to be followed through fluorescence techniques, but altered some properties of sTnC [Li et al. (1995) Biochemistry 34, 8330]. In the present study, direct calcium binding assays and fluorescence followed calcium titrations were used to better understand the effects of the Phe→Trp mutation at position 29 as well as the influence of each amino site on the calcium binding properties of the regulatory domain. Two new mutants were constructed in which the tryptophan analogs 5-hydroxytryptophan or 7-azatryptophan were introduced at position 29 (resulting in F29HW and F29ZW mutants, respectively). The data showed that when compared to sTnC, the Ca2+ affinity of amino sites was increased sixfold in F29W, nearly threefold in F29ZW and slightly decreased in F29HW. The F29ZW fluorescence followed Ca2+ titration displayed a bimodal curve that could be related to Ca2+ binding to each of the amino sites. This is the first report of fluorescence detection of the sequential Ca2+ binding to the regulatory sites. To investigate the influence of each amino site in the calcium binding or fluorescence properties of sTnC, F29W, F29HW and F29ZW, we have constructed double and triple mutants by replacing aspartate at position 30 or 66 (or both) by alanine. These mutations affect respectively the binding capacity of sites I and II. The data showed that: 1) in the calcium concentration range analyzed, the Asp→Ala mutation at position 30 impairs calcium binding to site I on1y, while Asp→Ala mutation at position 66 impairs calcium binding to both sites I and II, and 2) the Asp→Ala mutation at position 30 makes silent the replacement of Phe at position 29 by Trp, 5-hydroxytryptophan or 7-azatryptophan. We conclude that site I is essentially defunct without previous binding to site II and that position 29 influences the affinity of this adjusted site I.

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