This dissertation, studies two specific topics related to the research of computer-aided drug design(CADD) by employing the molecular simulations approach, that of protein drugs and that of small agents. These results can help drug designers to improve their products for treating special diseases. This work is divided into two parts: Protein drugs: Potential of mean force of the hepatitis C virus core protein¡Vmonoclonal 19D9D6 antibody interaction: Antigen-antibody interactions are critical for understanding antigen-antibody associations in immunology. To shed further light on this question, we studied a dissociation of the 19D9D6-HCV core protein antibody complex structure. However, forced separations in single molecule experiments are difficult, and therefore molecular simulation techniques were applied in our study. The stretching, that is, the distance between the centre of mass of the HCV core protein and the 19D9D6 antibody, has been studied using the potential of mean force calculations based on molecular dynamics and the explicit water model. Our simulations indicate that the 7 residues Gly70, Gly72, Gly134, Gly158, Glu219, Gln221 and Tyr314, the interaction region (antibody), and the 14 interprotein molecular hydrogen bonds might play important roles in the antigen-antibody interaction, and this finding may be useful for protein engineering of this antigen-antibody structure. In addition, the 3 residues Gly134, Gly158 and Tyr314 might be more important in the development of bioactive antibody analogues. Potential of mean force for syrian hamster prion epitope protein - monoclonal fab 3f4 antibody interaction studies: Simulating antigen-antibody interactions is crucial for understanding antigen-antibody associations in immunology. To shed further light into this question, we study a dissociation of syrian hamster prion epitope protein-fab3f4 antibody complex structure. The stretching (the distance between the center of mass of the prion epitope protein and the fab3f4 antibody) have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and implicit water model. For the complex structure, there are four important intermediates and two inter protein molecular hydrogen bonds in the stretching process. Inclusion of our simulations may help to understand the binding mechanics of the complex structure and will be an important consideration in design of antibodies against the prion disease. Potential of mean force for human lysozyme - camelid vhh hl6 antibody interaction studies: Calculating antigen-antibody interaction energies is crucial for understanding antigen-antibody associations in immunology. To shed further light into this equation, we study a separation of human lysozyme-camelid vhh hl6 antibody (cAb-HuL6) complex. The c-terminal end-to-end stretching of the lysozyme-antibody complex structures have been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) and explicit water model. For the lysozyme-antibody complex, there are six important intermediates in the c-terminal extensions process. Inclusion of our simulations may help to understand the binding mechanics of lysozym- cAb-HuL6 antibody complex. Small agents: Predictions of binding for dopamine D2 receptor antagonists by the SIE method: The control of tetralindiol derivative antagonists released through the inhibition of dopamine D2 receptors has been identified as a potential target for the treatment of schizophrenia. We employed molecular dynamics simulation techniques to identify the predicted D2 receptor structure. Homology models of the protein were developed on the basis of crystal structures of four receptor crystals. Compound docking revealed the possible binding mode. In addition, the docking analyses results indicate that five residues (Asp72, Val73, Cys76, Leu183, and Phe187) were responsible for the selectivity of the tetralindiol derivatives. Our molecular dynamics simulations were applied in combination with the solvated interaction energies (SIE) technique to predict the compounds' docking modes in the binding pocket of the D2 receptor. The simulations revealed satisfactory correlations between the calculated and experimental binding affinities of all seven tetralindiol derivative antagonists, as indicated by the obtained R2 value of 0.815. Combining homology modeling, docking, and molecular dynamics to predict the binding modes of oseltamivir, zanamivir, and Chinese natural herb products with the neuramindase of the H1N1 influenza A virus: The neuraminidase of the influenza virus is the target of the anti-flu drugs oseltamivir and zanamivir. Clinical practices show that zanamivir and oseltamivir are effective to treat the 2009 H1N1 influenza virus. Herein, we report the findings of molecular simulations for zanamivir, oseltamivir, and Chinese natural herb products with the neuramindase of the 2009 H1N1 influenza. Our approach theoretically suggests that the Glu278 residue is responsible for the neuramindase of the 2009 influenza drug selectivity.

Il formaggio sta dimostrando possedere, oltre alle caratteristiche nutrizionali classiche, degli aspetti nutrizionali innovati derivanti dalle proprietà di peptidi bioattivi contenuti nella frazione proteica caseinica del formaggio e rilasciati in seguito a proteolisi, ed ancora dalle proprietà di acidi grassi insaturi, quale l’acido linoleico coniugato. L’obiettivo di tale studio di dottorato è stato indagare su questi aspetti nutrizionali innovativi in formaggi tipici italiani quali Grana Padano e TrentinGrana, che pur essendo due formaggi molto simili tra loro, differiscono per l’uso della molecola antibatterica lisozima solo per la produzione del Grana Padano. Simulazioni in vitro della digestione gastrointestinale, nei campioni dei due formaggi a diversi tempi di stagionatura, ha dimostrato che esiste una relazione positiva tra tempo di stagionatura e digeribilità del calcio nel Grana Padano ,quando vengono considerati nella correlazione i risultati dei campioni con stagionatura superiore ai 24 mesi. A tempi di stagionatura inferiori a 24 mesi, i risultati di digeribilità del calcio del Grana Padano sono dispersi come appare nei campioni di TrentinGrana analizzati. Inoltre l’analisi RP-HPLC della distribuzione molecolare degli oligopeptidi dei campioni dei due formaggi, dimostra che la frazione peptidica coinvolta nel legame del calcio e quindi nel suo assorbimento, è quella compresa tra 1000 e 1500 D, e che l’analisi Seldi ha rilevato essere quella dei peptidi attivi fosfocaseinici. Inoltre differenze tra i due formaggi con e senza lisozima, appaiono solo per campioni tra 15 e 20 mesi di stagionatura, in cui campioni di formaggio senza lisozima appaiono più idrolizzati di quelli che lo contengono. Quindi dai risultati ottenuti appare che le differenze del profilo peptidico apportate dal lisozima non modificano le proprietà dei fosfopeptidi di assorbimento del calcio, forse influenzate da altri fattori che intervengono nella produzione del formaggio. L’attività ACE-inibitoria di abbassamento della pressione arteriosa esercitata da peptidi bioattivi è stata testata nei due formaggi, dimostrando che né il tempo di stagionatura e il grado di proteolisi, né il lisozima sono correlati all’attività ACE-inibitoria. Ed infine per l’importante ruolo che ha il minerale calcio nella dieta, indici di digeribilità del minerale in diversi alimenti sono stati considerati, in modo da poter formulare in maniera corretta diete alimentari coprendo il fabbisogno giornaliero con l’adeguato apporto energetico. I formaggi hanno dimostrato possedere indici di digeribilità del calcio superiori ad altri alimenti vegetali o a base di soia. In particolare il valore di digeribilità del Calcio del Grana Padano calcolato in vitro ha confermato il valore ottenuto in vivo , pari a 80%. / This study aimed to investigate about the not common known nutritional aspects of cheeses, which derive from their chemical components. In fact, in addition to the supply of macronutrient, cheeses are gaining interest as a source of bioactive peptides, of conjugated linoleic acid or for the new insight in the metabolic role of calcium. In vitro simulation of human gastrointestinal digestion revealed that cheeses have an higher digestibility of calcium than other foods, because of their casein-derived bioactive phosphopetides (CPPs) content has the ability to carry calcium minerals and avoid calcium precipitation, making it available for intestinal absorption. The in vitro calcium digestibility was calculated for different foods- cheeses, soya based foods and vegetables- to correct cover calcium requirements with an equilibrate energy intake. Calcium digestibility was also assessed in different ripened time Italian locally produced, semi fat, hard cheeses, Grana Padano and TrentinGrana. The main difference between them is the use or not of lysozyme during manufacturing. In Grana Padano samples, produced using lysozyme, there is a positive relationship between aging and dCa (r2 = 0.27; P<0.05) when sample > 24 months aged are considered (Grana Padano dCa results of samples less 24 months aged are quite widespread) , while in TrentinGrana, produced without the use of lysozyme, no significant correlation has been detected. RP-HPLC distribution analysis of oligopeptides molecular weight of these cheese showed that the only difference between them is that cheeses without lyzozyme, aged between 15 and 20 months, are more hydrolyzed than the same ripened time Grana Padano samples. Moreover the fraction of oligopeptides involved in calcium binding ranges between 1000 and 1500 D. SELDI analysis confirmed CPPs presence in this range. Therefore changes in cheese peptidic profiles probably caused by the use of lysozyme do not influence calcium digestibility because according to this study there is not a connection between change in peptidic profile and calcium digestibility results. The difference in calcium digestibility in Grana Padano samples aged over 24 month results should be probably ascribed also to the influence of other factors occurring during cheese manufacturing. Moreover, ACE-inhibitory activity of bioactive peptides was tested on in vitro digested Grana Padano and TrentinGrana samples with different ripening times. Correlation was not found between ACE-inhibitory activity and proteolysis level in different ripened time samples nor the lysozyme influence in releasing ACE-inhibitory bioactive peptides.

Thesis (Ph. D.)--University of Oregon, 2002. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 159-167). Also available for download via the World Wide Web; free to University of Oregon users.

A versatile AFM local oxidation lithography was developed for fabricating clean protein patterns ranging from nanometer to sub-millimeter scale on octadecyltrichlorosilane (OTS) layer of Si (100) wafer. This protein patterning method can generate bio-active protein pattern with a clean background without the need of the anti-fouling the surface or repetitive rinsing. As a model system, lysozyme protein patterns were investigated through their binding reactions with antibodies and aptamers by AFM. Polyclonal anti-lysozyme antibodies and anti-lysozyme aptamer are found to preferentially bind to the lysozyme molecules on the edge of a protein pattern before their binding to the interior ones. It was also demonstrated that the topography of the immobilized protein pattern affects the antibody binding direction. We found that the anti-lysozyme antibodies binding to the edge lysozyme molecules on the half-buried pattern started from the top but the binding on the extruded pattern started from the side because of their different spatial accessibility. In addition, after incubating lysozyme pattern with anti-lysozyme aptamer in buffer solution for enough long time, some fractal-shaped aptamer fibers with 1-6nm high and up to tens of micrometers long were formed by the self-assembling of aptamer molecules on the surface. The aptamer fibers anchor specifically on the edge of protein patterns, which originates from the biospecific recognition between the aptamer and its target protein. Once these edge-bound fibers have formed, they can serve as scaffolds for further assembly processes. We used these aptamer fibers as templates to fabricate palladium and streptavidin nanowires, which anchored on the pattern edges and never cross over or collapse over each other. The aptamer fiber scaffold potentially can lead to an effective means to fabricate and interface nanowires to existing surface patterns.

Structural biology is a branch of science related to biochemistry, biophysics, and molecular biology that deals with the molecular structures of biological macromolecules, in particular nucleic acids and proteins. Structure-guided drug design uses three-dimensional knowledge of protein structures to design small molecules which block the action of specific proteins. When crystals of theses macromolecules and their complexes can be obtained, their crystal structures can be determined by using isomorphous differences between a native structure and a derivative structure. This allows crystallographers to determine the coordinates of a small number of heavy atoms which provide initial phases for macromolecules. The advent of synchrotron radiation allowed determination of a heavy atom substructure by use of anomalous differences using either multiple wavelengths (MAD) or a single wavelength (SAD); the latter has become the most common phasing method in crystallography and is the method used in this study. The use of SeMet has been by far the most successful method employed in SAD. However, in some cases production of SeMet proteins is not possible thus necessitating additional options, for example, xenon. Noble gases such as xenon may be used in SAD experiments by binding to various, non-specific sites. Advances in noble gas pressurization systems like the Hampton Research Xenon Chamber have greatly eased the production of noble gas derivatives, xenon itself being a prime candidate with a very strong anomalous signal when compared to lighter noble gases like krypton and argon. Investigation of the phasing properties of xenon was carried out on test proteins hen egg white lysozyme (HEWL), thermolysin, glucose isomerase, and thaumatin II. Phases were successfully determined for all four proteins including thaumatin II which did not bind xenon but was successful due to the anomalous signal from 17 native sulfurs. The three remaining proteins showed varying occupancies and numbers of sites including xenon sites in thermolysin and glucose isomerase which have not been observed previously. This document will serve as a guide for the preparation of xenon derivative crystals and provides a strategy for the collection and processing of data from xenon derivatives.

Šiame darbe tirtas gramteigiamųjų ir gramneigiamųjų bakterijų atsakas į natyvaus ir katalitiškai neaktyvaus lizocimo bei jo 9 aminorūgščių katijoninio peptido sukeltus ląstelės apvalkalo pažeidimus. Panaudojant potenciometrinius metodus, buvo nustatyta, kad šie junginiai sukelia viduląstelinio K+ ištekėjimą iš L. lactis, B. subtilis ir P. aeruginosa ląstelių ir dalinę jų citoplazminės membranos depoliarizaciją, tačiau natyvus, kaitintas lizocimas ir 9a peptidas skirtingai veikia L. lactis apvalkalėlio laidumą. Peptidas bei kaitintas lizocimas sukelia staigesnį K+ jonų ištekėjimą ir membranos įtampos sumažėjimą nei natyvus lizocimas. Peptido sukeliamas K+ jonų ištekėjimas yra grįžtamas. Taip pat buvo įvertintas įvairių mutantinių L. lactis padermių jautrumas lizocimui. Pastebėta, kad kuo padermė atsparesnė lizocimui, tuo vėliau prasideda K+ jonų ištekėjimas iš šios padermės ląstelių. Pasitelkus DNR mikrogardelių metodą, buvo tiriamas lizocimo bei peptido poveikis L. lactis MG1363 bei &#916;oppA ląstelių genų raiškos pokyčiams. Nustatyta, jog peptidu ir lizocimu veiktose ląstelėse iššaukiama dvikomponentės valdymo sistemos CesSR raiška ir sukeliamas nuo SpxB priklausomas ląstelių atsparumas lizocimui. Gauti rezultatai rodo, jog lizocimas ir 9a peptidas sukelia kiek skirtingus L. lactis transkriptomo pokyčius: lizocimas savitai skatina nuo N-deacetilazės priklausomo atsparumo mechanizmo įsijungimą, taip pat skiriasi OpuA sistemą koduojančių genų raiška. / We used potenciometric measurments to investigate the response of Gram-positive and Gram-negative bacteria to cell envelope stress, caused by native and heat-inactivated lysozyme and lysozyme-derived 9 amino acid peptide. It was found that these antimicrobial compounds induce leakage of K+ outside the cells of L. lactis, B. subtilis and P. aeruginosa and cause partial depolarization of bacterial cytoplasmic membrane. We observed different response of L. lactis cells to these compounds – peptide and heat-inactivated lysozyme cause more rapid efflux of K+ ions than native lysozyme. Peptide has a reversible effect on K+ leakage. Sensitivity of different mutant strains of L. lactis to lysozyme was studied. It was shown that more resistant the strain is, the later the leakage of K+ ions is induced by lysozyme. To investigate the genome-wide response of L. lactis MG1363 and &#916;oppA strains to lysozyme and 9 a.a. peptide, changes of gene expression after challenging cells with these antimicrobial compounds were analysed using DNA microarrays. It was estimated, that lysozyme and lysozyme-derived 9a.a. peptide induce CesSR system and SpxB-mediated response. It was also shown that L. lactis response to 9a.a. peptide and lysozyme differs. Lysozyme specifically induces PgdA-mediated resistance mechanism. Changes of expression of OpuA system in lysozyme and peptide treated cells are also different.

Membrane chromatography was investigated as an alternative method to packed-bed chromatography for protein recovery. The purification of lysozyme from egg white with Natrix adseptTM weak cation-exchange membranes was investigated under two different binding configurations: (1) a non-flow, static set-up with variable pH and sodium chloride (NaCl) concentrations during the binding and elution steps, and (2) a dynamic, cross-flow set-up with recycle at pH 7.5 and no NaCl addition during binding. The weak cation-exchange membrane consisted of a carboxylic acid-based, environmentally-responsive hydrogel layer bonded to a polymer matrix. Lysozyme was chosen to illustrate protein-membrane binding interactions due to its well-characterized nature and positive surface charge over a large pH range. For the static binding set-up, two sources of lysozyme were studied: pure lysozyme and egg whites treated with 60 % (v/v) ethanol (ESEW). Elution of bound protein was performed with 1 M NaCl under two pH strategies: binding and elution at a constant pH, and binding at pH 4.5 and variable elution pH. The highest maximum total protein binding capacity for pure lysozyme and ESEW was observed at pH 4.5 with no NaCl addition; however, poor total protein and lysozyme activity recovery were achieved during separation. As well, other egg white proteins, such as ovomucoid, were observed to bind to the membrane surface at pH 4.5, despite possessing similar charge polarity to the anionic membrane surface, indicating a non-electrostatic binding mechanism during operation below the membrane’s pKa (4.7). Based on the conditions tested, the highest total protein and lysozyme activity recovery was demonstrated for the separation of lysozyme from ESEW at pH 7.5 binding and elution and no NaCl addition. In the dynamic binding study, very high pure lysozyme dynamic binding capacity was achieved at 10 % breakthrough (167.3 mg/ml membrane for a 0.35 mg/ml lysozyme solution). The lysozyme dynamic binding capacity was 2.2 times greater than the static binding capacity under similar conditions, significantly higher than published results for other cation-exchange membranes. The separation of lysozyme from four lysozyme sources was tested: pure lysozyme, ESEW, and aqueous egg whites with (ASEW) and without (AEW) 100 mM NaCl. The highest lysozyme activity recovery during separation and lysozyme purity was achieved from the ESEW feed. Lysozyme separation from aqueous egg whites was not as effective, likely due to a high concentration of negatively-charged protein impurities fouling the surface of the membrane. Competitive binding to the membrane limited lysozyme binding and reduced the purity of the recovery elution stream. The application of feed-side pressure during the separation of ESEW produced a high purity, high recovery lysozyme elution stream with a significant reduction in processing time; however, protein aggregates were observed to form on the membrane surface, limiting the applicability of high-pressure operation and reducing protein functionality in the elution stream. The weak cation-exchange membrane system was shown to successfully separate out a target protein from a low concentration protein mixture through electrostatic interactions, and may be further applied to other protein systems.

The extent of denaturation and glycation of lysozyme and BSA with the application of high hydrostatic pressure (HHP) at 400 MPa at 30°C from 8 to 48 hours and focused microwave irradiation at 50°C under varying microwave power and from 10 to 60 minutes was investigated in the presence and absence of D-glucose. The HHP treatment caused 10 to 20% denaturation of lysozyme whereas microwave irradiation caused 20 to 40% denaturation, with more destruction to the lysozyme in the presence of glucose compared to the control. The extent of glycation was also higher with the high pressure samples, causing 60% glycation upon 8 hours of high pressure exposure, but decreasing to around 40% thereafter. Microwave irradiation brought about 40% glycation to the lysozyme samples upon 20 min of irradiation. BSA, on the other hand, was more susceptible to damage by high energy exposures. BSA samples were denatured to a greater extent compared to lysozyme, up to 80% upon the prolonged exposures, but in all treatments, glucose seemed to act as a protectant contrary to the case of lysozyme. The extent of glycation detected was also minimal, ranging from 8 to 20%. / The feasibility of analyzing glycated proteins using pyrolysis-GC/MS was also investigated. Taking advantage of the formation of a diagnostic marker---2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one---upon pyrolysis of glycated proteins, the intensity of this peak was used to correlate the extent of glycation. The intensity of this peak in the pyrograms of glycated lysozymes was found to increase linearly with increasing incubation times and subsequently with the sugar loads of the glycated lysozyme. In addition, using the pyrograms as unique fingerprints, the extent of structural changes between modified and unmodified proteins were also assessed.

Thesis (M.S.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.

Submitted by (edna.saturno@ufrpe.br) on 2016-08-18T15:38:40Z No. of bitstreams: 1 Claudio Henrique Clemente Fernandes.pdf: 1914599 bytes, checksum: da7b1a519963cc8ffa30d5c1998aa94f (MD5) / Made available in DSpace on 2016-08-18T15:38:40Z (GMT). No. of bitstreams: 1 Claudio Henrique Clemente Fernandes.pdf: 1914599 bytes, checksum: da7b1a519963cc8ffa30d5c1998aa94f (MD5) Previous issue date: 2007-02-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The bovine enzootic leucosis (BEL) is a contagious infect disease cosmopolitan, with immune depressor potential, characterized by chronic evolution and great injuries that cause to the national cattle- breeding. The general objective of the achievement of this study was to contribute to the Bovine Leukosis Virus (BLV), realizing immune serologic essay to find dimension of the infection (serum-prevalent) and establish a parameter for evaluation of the immune condition (levels of serum lysozyme) of milky cattle breeding raised in the north region of Tocantins state. It was colleted serum samples of 38 bovine herds, totalizing 881 samples submitted to the double radial immune diffusion test of Ouchterlony in agar gel. From this universe of samples, 400 samples were divided in 2 groups. According to the immune serologic result obtained. G1 200 BLV negative samples, G2 BLV positive samples. The samples were still submitted to a simple radial immunodiffusion of Mancini to define the serum concentration of lysozyme which obtained results were statistically analyzed by t- student test. The results demonstrated that the BEL is amply disseminated in bovine herds examined (94,7% - 36/38), with a prevalence of serum antibodies anti BLV of the studied population corresponding to 37% (328/821) this is the first register of the infection in Tocantins State. The serum levels of lysozyme found G1 8,94 /ml e G2 7,05 /ml, knowing that the average difference between the two groups was 1,92 /ml, so, more elevated in the BLV negative animals than in the BLV positive ones, this revealed how significant is that difference. It can signalize an immune depression state of the immunity system. The obtained results demonstrated the large dissemination of BEL, being the influence, of risk factors in connection with a reduction of levels of lysozyme in animals BLV positives, and suggest that immune serologic tests realized, make possible the validation of an important parameter of evaluation of immune state of BLV positive bovine contributing, this way, to elucidation of immune depressor paper of bovine leucosis virus. / A Leucose Enzoótica dos Bovinos (LEB) é uma doença infecto-contagiosa, cosmopolita, de potencial imunodepressor, caracterizada pela evolução crônica e pelos grandes prejuízos que determina a pecuária bovina nacional. O objetivo geral com a realização deste estudo foi contribuir para a elucidação do papel imunodepressor do Vírus da Leucose Bovina (VLB), realizando ensaios imunossorológicos para dimensionar a infecção (soroprevalência) e estabelecer um parâmetro de avaliação do estado imunitário (teores de lisozima sérica) de bovinos leiteiros criados na Região Norte do Estado do Tocantins. Foram colhidas amostras séricas de 38 rebanhos bovinos, totalizando 882 amostras submetidas ao teste de Imunodifusão Radial Dupla de Ouchterlony em gel de ágar. Desse universo amostral, 400 amostras foram divididas em dois grupos, em função do resultado imunossorológico obtido: G1 200 amostras VLB negativos e G2 200 amostras VLB positivos. As amostras foram submetidas, ainda, à Imunodifusão Radial Simples de Mancini para determinar a concentração sérica da lisozima, cujos resultados obtidos foram analisados estatisticamente pelo teste t-Student. Os resultados demonstraram que a LEB encontra-se amplamente disseminada nos rebanhos examinados (94,7% - 36/38), com uma prevalência de anticorpos séricos anti-VLB da população estudada igual a 37,0% (326/881), sendo este o primeiro registro da infecção no Estado do Tocantins. Os níveis séricos de lisozima encontrados foram G1 8,94 g/ml e G2 7,05 g/ml, sendo que a diferença média entre os dois grupos foi de 1,92 g/ml, portanto, mais elevado nos animais VLB negativos do que nos VLB positivos, diferença esta que se revelou significante, sinalizando um estado imunitário de imunodepressão. Os resultados obtidos demonstraram a ampla disseminação da LEB, sob a influência de fatores de risco, em conexão com a redução dos teores de lisozima nos animais VLB positivos, e sugerem que os ensaios imunossorológicos realizados possibilitaram a validação de um importante parâmetro de avaliação do estado imunitário de bovinos VLB positivos, contribuindo, desta forma, para a elucidação do papel imunodepressor do Vírus da Leucose Bovina.