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Water has long been assumed to be essential for biological function. To understand the molecular basis of the role of water in protein function, several studies have established a correlation between enzyme activity and hydration level. While a threshold of hydration of 0.2 h (grams of water per gram of dried protein) is usually accepted for the onset of enzyme activity, recent works show that enzyme activity is possible at water contents as low as 0.03 h (Lind et al., 2004). Diffusion limitation in these experiments was avoided by monitoring enzyme-catalyzed hydrolysis of gas-phase esters. However, since water is also a substrate for the enzyme used in these experiments, they cannot be used to probe the possibility of activity at zero hydration. However, the pig liver esterase and C. rugosa lipase B are able to catalyse alcoholysis reactions in which an acyl group is transferred between an ester and an alcohol. Therefore, by following this reaction and using a gas phase catalytic system, we have been able to show that activity can occur at 0 g/g. These results led to the question of the accuracy of determinations of very low water concentrations; i.e., how dry is 0 g/g? Although gravimetric measurements of the hydration level do not allow us to define the anhydrous state of the protein with sufficient sensitivity, using 18O-labeled water, we have been able to quantify the small number of water molecules bound to the protein after drying, using a modification of the method of Dolman et al. (1997). Testing different drying methods, we have been able to determine a level of hydration as low as 2 moles of water per mole of protein (equivalent to 0.0006 h in the case of pig liver esterase) and have shown that in the case of the pig liver esterase, activity can occur at this hydration level. At the molecular level, if the hydration level affects activity, we can expect an effect on the protein dynamics. Neutron scattering spectra of hydrated powders, for instance, show that diffusive motions of the protein increase with the hydration (Kurkal et al., 2005) To address the question of the protein motions involved in the onset of enzyme activity at low hydration, we performed neutron scattering experiments on a pico-second time scale on dried powders. Preliminary results show a dynamical transition at hydration levels as low as 3 h. Molecular dynamic simulations have also been used in this study to access the dynamics of the active site. Overall, the results here show that pig liver esterase can function at zero hydration, or as close to zero hydration as current methods allow us to determine. Since the experimental methodology restricts this work to a small number of enzymes, it is unlikely that it will ever be possible to determine if all enzymes can function in the anhydrous state: however, the results here indicate that water is not an obligatory requirement for enzyme function.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel provides the principal uptake pathway for protons. A water chain is thought to mediate the relay of protons through the D-channel, but it is interrupted at N139 in all crystallographic structures. Here, free energy simulations are used to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, where reduction and pumping is compromised. A general approach for the calculation of water occupancy in protein cavities is presented and demonstrates that combining efficient sampling algorithms with long simulation times is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of conformational and hydration states of the D-channel is characterized. Results shed light onto the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes.

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<div>Deep neural networks (DNNs) have gained tremendous attention over the recent years due to their outstanding performance in solving many problems in different fields of science and technology. Currently, this field is of interest to many researchers and growing rapidly. The ability of DNNs to learn new concepts with minimal instructions facilitates applying current DNN-based methods to new problems. Here in this dissertation, three methods based on DNNs are discussed, tackling different problems in the field of computer-aided drug design.</div><div><br></div><div>The first method described addresses the problem of prediction of hydration properties from 3D structures of proteins without requiring molecular dynamics simulations. Water plays a major role in protein-ligand interactions and identifying (de)solvation contributions of water molecules can assist drug design. Two different model architectures are presented for the prediction the hydration information of proteins. The performance of the methods are compared with other conventional methods and experimental data. In addition, their applications in ligand optimization and pose prediction is shown.</div><div><br></div><div>The design of de novo molecules has always been of interest in the field of drug discovery. The second method describes a generative model that learns to derive features from protein sequences to design de novo compounds. We show how the model can be used to generate molecules similar to the known for the targets the model have not seen before and compare with benchmark generative models.</div><div><br></div><div>Finally, it is demonstrated how DNNs can learn to predict secondary structure propensity values derived from NMR ensembles. Secondary structure propensities are important in identifying flexible regions in proteins. Protein flexibility has a major role in drug-protein binding, and identifying such regions can assist in development of methods for ligand binding prediction. The prediction performance of the method is shown for several proteins with two or more known secondary structure conformations.</div>

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Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-07-25T13:32:13Z No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-26T12:05:00Z (GMT) No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-26T12:05:00Z (GMT). No. of bitstreams: 2 Dissertação - Junaine Vasques da Silva - 2002.pdf: 3727327 bytes, checksum: 4cb8c1db4d3fb95798779f39aae78673 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2002-12-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The main function of the most superficial layer of the epidermis, the Stratum Corneum (SC), is to provide a physical barrier that controls the transepidermal water loss as well as the permeation of another substances in both directions across the skin. The SC is formed by anabolically dead cells, the terminally differentiated corneocyte, and its function is essentially accomplished by forming a highly insoluble protein structure on the surface of the corneocytes, termed the cornified cell envelope, and by impeding water diffusion across the SC by mortaring the corneocytes together by layers of skin-specific lipids, essentially ceramide, cholesterol and fatty acid. In this work the cell envelope of the SC was spin labeled with a sulfhydryl-specific nitroxide reagent to investigate the water content effects upon the protein dynamics directly in the intact tissue. A two-state model for the nitroxide side chain described the coexistence of two spectral components in the electron paramagnetic resonance (EPR) spectra. The so-called strongly immobilized component, S, is associated with the EPR signal of a motionally restricted nitroxide fraction having its N-O group hydrogen bonded to protein (rigid structure) while the weakly immobilized component, W, corresponds to the signal provided by the spin labels with higher mobility (~10 times greater) exposed to the aqueous environment. The relative populations between these two mobility states, S and W, are in thermodynamic equilibrium. The standard Gibbs free energy, enthalpy and entropy changes for transferring the nitroxide side chain from the state contacting the solvent, W, to the one contacting protein, S, indicated that the reduction of the SC water content to below ~h 0.69, g H2O per g dry SC, stabilizes the protein interacting state, S. Upon decreasing the SC hydration level below ~h 0.69 the segmental motion of the polypeptide chains and the rotational motion of the spin-labeled side chain were also constrained. To test our methodology in a pure and very well known protein, we also studied the effects of two types of detergents on the bovine serum albumin (BSA). Both detergents, the anionic sodium dodecyl sulfate (SDS) and the zwitterionic N-hexadecyl-N,N-dimethyl-3-ammonium-1-propanesulfonate (HPS) increase the mobility of the protein backbone and of the nitroxide side chain. The thermodynamic parameters indicated that these detergents destabilize the protein favoring less compact conformations. This work can also be useful to improve the spectral analysis of site-directed spin labeling, especially for a more quantitative description in terms of thermodynamic parameters. / A camada mais superficial da epiderme, o Estrato Córneo (EC), tem como função principal a formação de uma barreira física que controla a perda de água do corpo bem como a permeação de outras substâncias em ambas as direções da pele. O EC é formado por células anabolicamente mortas, os corneócitos, os quais sofreram diferenciação celular terminal, e sua função é realizada formando uma estrutura de proteínas altamente insolúveis na superfície do corneócito, chamada de envelope celular, e também uma matriz lipídica, essencialmente ceramídios, colesterol e ácidos graxos, que dificultam a difusão da água. Neste trabalho, o EC foi marcado com marcadores de spin específicos para reagir com os grupos sulfidrilas das proteínas, para investigar os efeitos do conteúdo de água na dinâmica de proteínas diretamente no tecido intacto. Um modelo de dois estados para a cadeia lateral do nitróxido descreveu a coexistência de duas componentes espectrais de ressonância paramagnética eletrônica (RPE). A componente denominada fortemente imobilizada (S), surge de uma fração de marcadores com o átomo de oxigênio do nitróxido ligado à proteína (estrutura rígida) enquanto a componente fracamente imobilizada é gerada pelos marcadores com mobilidade mais alta (~10 vezes maior) e expostos ao ambiente aquoso. As populações relativas entre estes dois estados de mobilidade, S e W, estão em equilíbrio termodinâmico. Os parâmetros da termodinâmica: energia livre padrão de Gibbs, entalpia e entropia, envolvidos na transferência da cadeia lateral do nitróxido do estado W, contatando ao solvente, para o estado S, contatando a proteína, indicaram que a redução do conteúdo de água para abaixo de ~0.69g de H2O por g de EC seco, estabiliza o estado S (cadeia lateral do nitróxido dobrada sobre a cadeia principal da proteína). Ao diminuir o nível de hidratação para abaixo de ~ h 0.69 (g H2o/g EC seco) o movimento local da cadeia polipeptídica e o movimento rotacional da cadeia lateral do marcador de spin foram ambos reduzidos. Para testar nossa metodologia em uma proteína pura e bem conhecida, estudamos os efeitos de dois tipos de detergentes sobre a albumina do soro bovino (BSA). Ambos os detergentes, o aniônico dodecil sulfato de sódio (SDS) e o ziteriônico N-hexadecil-N,N-dimetil-3-amônio-1-propanosulfonato (HPS) aumentaram a mobilidade da cadeia principal da proteína e da cadeia lateral do nitróxido. Os parâmetros termodinâmicos indicaram que estes detergentes desestabilizam a proteína favorecendo conformações menos compactas. Os resultados do presente trabalho também podem contribuir para aprimorar a