In this thesis, the structure and stability of globular proteins adsorbed onto nanometer-sized hydrophilic silica particles were investigated using differential scanning calorimetry (DSC), hydrogen/deuterium exchange (HDX), and mass spectrometry (MS). The adsorption process itself was characterized with fluorescence and absorption spectroscopy and surface plasmon resonance (SPR). The combination of these methods offered a unique insight into adsorption-induced changes within proteins related to their adsorption characteristics. DSC contributed with thermodynamic information on the overall structural stability within the protein population. HDX in combination with MS contributed information on the structure and stability of adsorbed proteins with focus on changes within the secondary structure elements. In order to increase the structural resolution in this part of the investigation, proteolysis was performed prior to the MS analyzing step. Knowledge on the protein adsorption process was utilized in a practical approach called ligand fishing. In this approach, SPR was used to monitor the chip-based affinity purification of a protein with MS used for protein identification. Adsorption isotherms revealed that electrostatic interactions play an important role in the adsorption of proteins to hydrophilic surfaces. DSC investigation revealed that the thermal stability of proteins reduces with increasing electrostatic attraction between the protein and the surface and that this effect diminishes at higher surface coverage. The mass-increase due to exchange between protein hydrogen atoms and deuterium atoms in solution was investigated as a function of time. This gave insight into adsorption-induced changes in the structural stability of proteins. By combining DSC and HDX-MS, it was possible to differentiate between adsorption-induced changes in the secondary and tertiary structure. Additionally, if limited proteolysis was performed, the investigations gave insight into the orientation and protein segment specific changes in the stability of proteins adsorbed to silica surfaces. The adsorption of proteins to silica particles also provided the basis for a new experimental design that allows handling of minute amounts of proteins in a ligand fishing application, as used in the field of functional proteomics.

Xylanases are xylan-degrading enzymes, belong to glycosyl hydrolases (GH). Xylanases from the two major families 10 (GH10) and 11 (GH11) catalyze the hydrolysis of internal β-, bonds of xylan backbone. Xylan is the second most abundant polysaccharide in nature. Nearly one third of the dry weight of the higher plants is xylan and therefore, xylanases have an important role in biomass conversions. Currently, the most effective application of xylanases is in prebleaching of kraft pulp to minimize the use of environmentally hazardous chemicals in the subsequent treatment stages. In recent years, therefore, attention is focused on to isolate and/or engineer the xylanases for the industrial requirements. The desirable roperties of xylanases in paper industry are stability and activity at high temperatures and alkaline pH. While he factors responsible for the thermal stability of GH10 xylanases have been analyzed, factors governing the alkaline stability of GH10 xylanases remain poorly understood. The present thesis reports the crystal structures of an alkali thermostable GH10 extracellular endo-xylanase (BSX) from an alkalophilic organism, Bacillus sp. NG-27 in free and xylosaccharides-bound form. The enzyme was purified from the native organism and crystallized. The structure was solved by molecular replacement method. The 2.2 Å crystal structure of the native BSX enzyme is the first structure of an alkali thermostable GH10 family xylanase from an alkalophilic organism. It has unveiled unique protein properties that can form the basis for improving the thermal, alkaline stability and activity by site directed mutagenesis. The comparative study, especially in relation to GH10 xylanases, deciphered important structural features which are likely to be responsible for the alkaline stability of the enzyme. The work exemplifies the mechanism of adaptation of enzymes to function under polyextreme conditions through changes in the nature and composition of solvent-exposed residues. As apparent from the comparative study, the enhanced stability of the protein can be attributed to the surface rich in acidic residues and less number of solvent-exposed Asn as seen in BSX. This situation which may be roughly described as “acidic residues outside and Asn inside”, is a notable feature of alkali-stable GH10 xylanases from alkalophilic organisms. In addition, the candidate has carried out the comprehensive database analysis of the occurrence of C-H…O hydrogen bonds in helices and helix termini of globular proteins. The study provides a compelling evidence that the main-chain Cα and the side-chains CH which participate in C-H…O hydrogen bonds collectively augment the cohesive energy and thereby contribute together with the classical N-H…O hydrogen bonds and other interactions to the overall stability of helix and therefore of proteins. Chapter 1 starts with a brief introduction of xylanases, their classifications and overall folds. At present, a little more than a dozen of crystal structures of GH10 xylanases are known and described in the literature. A brief mention about these structures and their optimum pH and temperature is outlined under a separate section. In view of the industrial importance of the study enzyme, the potential industrial and biotechnological applications of xylanases are detailed in this Chapter. A section is dedicated to describe the present study enzyme BSX, an alkali thermostable endo-xylanase from an alkalophilic bacterium, Bacillus sp. NG-27. BSX has a molecular mass of ~41 kDa and is optimally active at 343 K and at a pH of 8.4. The alkaline thermostability of the wild type BSX is likely to be industrially important. At the end, the scope of the present work is detailed. Chapter 2 presents the purification of xylanase (BSX) from Bacillus sp. NG-27, the crystallization of the native and xylosaccharides-bound BSX, the X-ray diffraction data collection on these crystals and processing of the data. Repeated attempts to crystallize the protein expressed in the chloroplast of transgenic tobacco plant were unsuccessful. However, crystallization was achieved with the protein sample purified from the native source by hanging drop vapour diffusion method. Crystals were grown at both acidic (4.6) and basic pH (8.5). The corresponding crystallization conditions are 0.2 M MgCl2, 0.1 M sodium acetate pH 4.6 and 20% PEG 550 MME and 0.1 M aCl, 0.01 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000. Crystals grown at acidic pH were not suitable for X-ray diffraction study. Subsequently, crystal obtained at a basic pH of 8.5 was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). Native BSX crystals belong to monoclinic space group C2 with unit cell parameters a = 174.5 Å, b = 54.7 Å, c = 131.5 Å and β = 131.2°. Crystals of xylosaccharides-bound enzyme were grown in a slightly modified crystallization condition of native, 0.1 M NaCl, 0.2 M MgCl2, 0.1 M Tris-HCl pH 8.5 and 15% PEG 8000 and the enzyme was incubated with xylan prior to setting up the crystallization. Crystals belong to primitive orthorhombic space group P212121 with unit cell parameters a = 59.2 Å, b = 83.8 Å and c = 174.4 Å. A data set was collected using synchrotron radiation of wave length 1.0 Å from a cryo-cooled crystal at Spring-8 BL26B1 beam line, Japan. The Matthews coefficient VM for native and xylosaccharides- bound crystals was calculated to be 2.8 and 2.7 Å3 Da-1, respectively, suggesting two molecules in each crystal asymmetric unit. No twinning was detected in both the datasets and the overall quality of the data sets was found to be good. Chapter 3 details the application of molecular replacement method to the structure solution of native and xylosaccharides-bound BSX, the course of iterative model building and the refinement carried out, and the quality of the final protein structure models. The native-enzyme structure solution was obtained by the molecular replacement method using as a search model the crystal structure (PDB code 1hiz) of the closest homologous, extracellular xylanase (GSX) from Geobacillus stearothermophilus. No non- crystallographic symmetry (NCS) restraint was applied between the two independent molecules in the crystal asymmetric unit at the final round of refinement. The final positional refinement of native BSX converged to R factors of R = 19.4% and Rfree = 23.5% for data between 20.0 to 2.2 Å. The final native model consists of 5704 protein atoms, two Mg2+ ions and 721 solvent water molecules. The final native model was taken as the search structure for the xylosaccharides-bound BSX and a solution with a correlation coefficient of 70.7% and an R-factor of 32.1% was obtained from the molecular replacement calculation. Unlike the native structure refinement, NCS restraint was imposed at all stages of the refinement. Bound xylosaccharides were clearly visible inthe difference Fourier electron density maps. The last round of refinement gave a model with R and Rfree of 21.8% and 25.7%, respectively. The final xylosaccharides-bound model consists of 5766 protein atoms, four Mg2+ ions, 85 atoms belong to bound xylosaccharides and 523 solvent water molecules. No residues were found in the disallowed region of the Ramachandran (φ, ψ) map for both the structures. Chapter 4 describes the native and xylosaccharides-bound BSX crystal structures and the structural comparison of BSX with other GH10 family xylanase crystal structures for which the optimum temperature and pH are known in the literature. BSX folds as the ubiquitous (β/α)8-barrel, a common structural superfold characteristic of GH10 xylanases. The two active site glutamic acid residues, Glu149 and Glu259, are located on opposite sides of the active site cleft and their side-chains are at a distance of 5.5 Å apart suggesting the enzymatic reaction takes place by the retaining mechanism. From the structural superposition of other xylotriose-bound xylanase structures on to the xylosaccharides-bound BSX, structural plasticity in the xylotriose binding can be inferred, implying that the xylose recognition at the subsite -3 displays plasticity and is less specific as opposed to that at -1 and -2 subsites. The stacking interaction of one of the xylose moieties of the xylobiose with the Trp235 seen in BSX provides, for the first time, a structural evidence for the direct involvement of Trp235 in xylosaccharides binding. The crystal structure revealed a metal binding site, found at the C-terminal end of catalytic domain. The presence of metal binding site was not anticipated from earlier theoretically modeled structure and biochemical studies. Further, we have shown experimentally the requirement of Mg2+ ion for the enzyme activity. We havedescribed a novel WP sequence-structure-interaction motif which is present in the (+) side of the active site region and presumably helps in the efficient binding of the carbohydrate moiety of the xylan in the active site cleft of BSX. The structural comparison of BSX with other GH10 xylanases solved to date and characterized to be active at a pH close to neutral was done for the first time. The comparative study revealed the essential structural features which may responsible for the alkaline stability of GH10 xylanases.Briefly, the alkalophilic GH10 xylanases from alkalophilic organisms have surface abundant in acidic residues, the heat and alkaline susceptible residue Asn depleted on the protein surface and increased number of salt bridges. Our study has unveiled the role of the nature and composition of protein surface amino acids in the adaptation of enzymes to polyextreme conditions. The observations reported in the thesis provide important lessons for engineering alkaline stability in xylanases for industrial applications and in general for the understanding of alkaline stability in related proteins. A comparison of the surface features of the BSX and of halophilic proteins allowed us to predict the activity of BSX at high salt concentrations, which we verified through experiments. This offered us important lessons in polyextremophilicity of proteins, where understanding structural features of a protein stable in one set of extreme conditions provided clues about the activity of the protein in other extreme conditions. Chapter 5 summaries the important findings of the present study from the crystal structural analysis of BSX and its comparison with non-alkalophilic GH10 xylanases. Separate sections are made on conclusions and future prospects for the study on BSX. Chapter 6 describes the comprehensive database analysis of C-H…O hydrogen bond in helices of globular proteins. The C-H…O hydrogen bonds found in helices are predominantly of type 5 → 1 or 4 → 1.Our analysis reveals that the Cγ and Cβ hydrogen atom(s) are frequently involved in such hydrogen bonds. A marked preference is noticed for aliphatic β-branched residue Ile to participate in 5 → 1 C- H…O hydrogen bonds involving methylene Cγ1 atom as donor in α-helices. In addition, C-H…O hydrogen bonds are present along with helix stabilizing salt bridges and to some extent compensate for the side-chain conformational entropy loss. Our analysis highlights that a multitude of local C-H…O hydrogen bondsformed by a variety of amino acid side-chains and Cα hydrogen atoms occur in helices and more so at the helix termini. A majority of the helix favouring residues, Met, Glu, Arg, Lys, Leu and Gln which also have large side-chains with more donatable CH groups, have significant propensity to form side-chain to main-chain C-H…O hydrogen bonds in helix. The large side-chains are marked by their ability to shield from the solvent the polar atoms of the peptide backbone and at the same time participate in weak cohesive C-H…O interactions in the helix. This chapter also details the identification for the first time a novel chain reversal motif stabilized by 1 → 5 Cα-H…O interactions. The importance of these hydrogen bonds with respect to helix stability is discussed in the final section of the chapter. Appendix A details the crystallographic and structural analyses oftwares used for the present thesis work. Appendix B describes, in addition to the crystal structure analysis of BSX, the work carried out by the candidate on a comparative study of a thermostable xylanase from Thermoascus aurantiacus, solved in our laboratory at atomic 1.11 Å (293 K) and ultrahigh 0.89 Å (100 K) resolutions. From the comparison, we have for the first time pointed out the possibility of plasticity of ion pairs in proteins with water molecules mediating some of the alternate arrangements. The αβ-loops are relatively less flexible than the βα-loops. The β-strands are least affected structurally with the increase in temperature. Thus the TIM barrel fold in the study enzyme, though having a single domain, may be dissected into parts based on the relative flexibility and described as having a rigid core constituted by the β-barrel and a less rigid exterior formed by the surrounding α-helices. Appendix C presents the crystallization and the preliminary X-ray characterization work done by the author of the thesis on an alkali thermostable cellulase enzyme from Thermomonospora sp. The protein is an extracellular enzyme with molecular mass of 14.2 kDa and interestingly, has the dual activity for both cellulose and xylan. The primary structure of the enzyme is not known. The enzyme was purified from the source organism and crystallized. A complete diffraction data set was collected and processed to 2.3 Å in an orthorhombic space group P212121. Appendix D contains tables which give details about the analysed 5 → 1 Cα- H…O hydrogen bonds in helices and a novel chain reversal motif with 1 → 5 Cα-H…O hydrogen bonds. Appendix E encloses reprints of publications which have resulted from the work reported in the thesis.

Globular clusters are dense and massive star clusters that are an integral part of any major galaxy. Careful studies of their stars, a single cluster may contain several millions of them, have revealed that the ages of many globular clusters are comparable to the age of the Universe. These remarkable ages make them valuable probes for the exploration of structure formation in the early universe or the assembly of our own galaxy, the Milky Way. A topic of current research relates to the question whether globular clusters harbour massive black holes in their centres. These black holes would bridge the gap from stellar mass black holes, that represent the final stage in the evolution of massive stars, to supermassive ones that reside in the centres of galaxies. For this reason, they are referred to as intermediate-mass black holes. The most reliable method to detect and to weigh a black hole is to study the motion of stars inside its sphere of influence. The measurement of Doppler shifts via spectroscopy allows one to carry out such dynamical studies. However, spectroscopic observations in dense stellar fields such as Galactic globular clusters are challenging. As a consequence of diffraction processes in the atmosphere and the finite resolution of a telescope, observed stars have a finite width characterized by the point spread function (PSF), hence they appear blended in crowded stellar fields. Classical spectroscopy does not preserve any spatial information, therefore it is impossible to separate the spectra of blended stars and to measure their velocities. Yet methods have been developed to perform imaging spectroscopy. One of those methods is integral field spectroscopy. In the course of this work, the first systematic study on the potential of integral field spectroscopy in the analysis of dense stellar fields is carried out. To this aim, a method is developed to reconstruct the PSF from the observed data and to use this information to extract the stellar spectra. Based on dedicated simulations, predictions are made on the number of stellar spectra that can be extracted from a given data set and the quality of those spectra. Furthermore, the influence of uncertainties in the recovered PSF on the extracted spectra are quantified. The results clearly show that compared to traditional approaches, this method makes a significantly larger number of stars accessible to a spectroscopic analysis. This systematic study goes hand in hand with the development of a software package to automatize the individual steps of the data analysis. It is applied to data of three Galactic globular clusters, M3, M13, and M92. The data have been observed with the PMAS integral field spectrograph at the Calar Alto observatory with the aim to constrain the presence of intermediate-mass black holes in the centres of the clusters. The application of the new analysis method yields samples of about 80 stars per cluster. These are by far the largest spectroscopic samples that have so far been obtained in the centre of any of the three clusters. In the course of the further analysis, Jeans models are calculated for each cluster that predict the velocity dispersion based on an assumed mass distribution inside the cluster. The comparison to the observed velocities of the stars shows that in none of the three clusters, a massive black hole is required to explain the observed kinematics. Instead, the observations rule out any black hole in M13 with a mass higher than 13000 solar masses at the 99.7% level. For the other two clusters, this limit is at significantly lower masses, namely 2500 solar masses in M3 and 2000 solar masses in M92. In M92, it is possible to lower this limit even further by a combined analysis of the extracted stars and the unresolved stellar component. This component consists of the numerous stars in the cluster that appear unresolved in the integral field data. The final limit of 1300 solar masses is the lowest limit obtained so far for a massive globular cluster. / Kugelsternhaufen sind dichte, gravitativ gebundene Ansammlungen von teilweise mehreren Millionen Sternen, die ein fester Bestandteil jeder massiven Galaxie sind. Aus der Untersuchung der Kugelsternhaufen in der Milchstraße weiß man, dass das Alter von vielen dieser Objekte vergleichbar ist mit jenem des Universums. Dies macht sie zu wertvollen Forschungsobjekten, beispielsweise um die Entstehung der Milchstraße und die Strukturbildung im frühen Universum zu verstehen. Eine aktuelle wissenschaftliche Fragestellung befasst sich damit, ob Kugelsternhaufen massive schwarze Löcher beherbergen. Diese würden eine Brücke schlagen von den stellaren schwarzen Löchern, die durch den Kollaps massereicher Sterne entstehen, zu den supermassiven schwarzen Löchern, welche man in den Zentren massiver Galaxien beobachtet. Man bezeichnet sie daher auch als mittelschwere schwarze Löcher. Die sicherste Diagnostik, um schwarze Löcher zu detektieren und ihre Masse zu bestimmen ist, die Bewegung der Sterne innerhalb ihrer gravitativen Einflusssphäre zu vermessen. Spektroskopische Untersuchungen vermögen dies über die Dopplerverschiebung von Spektrallinien, sind jedoch in dichten stellaren Feldern wie Kugelsternhaufen schwierig. Aufgrund der Turbulenz in der Atmosphäre und dem endlichen Auflösungsvermögen des Teleskops erscheinen die Sterne in den Beobachtungen nicht punktförmig, sondern mit einer durch die Punktspreizfunktion (PSF) gegebenen Breite. In dichten stellaren Feldern führt dies dazu, dass die Sterne überlappen. Da klassische spektroskopische Verfahren nicht bildgebend sind, lassen sich die Beiträge der Einzelsterne zu einem beobachteten Spektrum nicht trennen und die Geschwindigkeiten der Sterne können nicht vermessen werden. Bildgebende spektroskopische Verfahren, wie etwa die Integralfeld-Spektroskopie, bieten jedoch die Möglichkeit, die PSF zu rekonstruieren und basierend darauf die Spektren überlappender Sterne zu trennen. Im Rahmen der vorgelegten Arbeit wird das Potential der Integralfeld-Spektroskopie in der Beobachtung dichter stellarer Felder zum ersten Mal systematisch analysiert. Hierzu wird eine Methodik entwickelt, die das Extrahieren von Einzelsternspektren über eine Rekonstruktion der PSF aus den vorhandenen Daten erlaubt. Anhand von Simulationen werden Voraussagen darüber gemacht, wie viele Sternspektren aus einem gegebenen Datensatz extrahiert werden können, welche Qualität diese Spektren haben und wie sich Ungenauigkeiten in der rekonstruierten PSF auf die Analyse auswirken. Es zeigt sich hierbei, dass die entwickelte Methodik die spektroskopische Analyse von deutlich mehr Sternen erlaubt als klassische Verfahren. Parallel zu dieser systematischen Studie erfolgt die Entwicklung einer dezidierten Analysesoftware, welche im zweiten Teil der Arbeit auf Daten von drei Kugelsternhaufen angewendet wird, die mit dem PMAS Integralfeld-Spektrographen am Calar Alto Observatorium aufgenommen wurden: M3, M13 und M92. Die Auswertung dieser Daten liefert Spektren für eine Stichprobe von ungefähr 80 Sternen pro Kugelsternhaufen, weit mehr als bisher im Zentrum eines der drei Haufen verfügbar waren. In der weiteren Analyse werden Jeans Modelle für jedes der drei Objekte gerechnet. Diese erlauben basierend auf einer angenommenen Massenverteilung innerhalb des Kugelsternhaufens eine Vorhersage der Geschwindigkeitsdispersion der Sterne. Der Vergleich mit den gemessenen Geschwindigkeiten zeigt, dass in keinem der drei Haufen ein schwarzes Loch benötigt wird, um die Dynamik der zentrumsnahen Sterne zu erklären. Im Gegenteil, die Beobachtungen können zu 99,7-prozentiger Sicherheit ausschließen, dass sich in M13 ein schwarzes Loch mit einer Masse größer 13000 Sonnenmassen befindet. In den anderen beiden Haufen liegt diese Grenze noch bei deutlich geringeren Massen, nämlich bei 2500 Sonnenmassen in M3 und 2000 Sonnenmassen in M92. In M92 ist es außerdem möglich, das Limit noch weiter herabzusetzen durch eine zusätzliche Analyse der unaufgelösten stellaren Komponente. Diese Komponente besteht aus dem integrierten Licht all jener Sterne, die zu schwach und zahlreich sind als dass sie aus den verfügbaren Daten einzeln extrahiert werden könnten. Das endgültige Limit von 1300 Sonnenmassen ist das geringste, welches bisher in einem massiven Kugelsternhaufen gemessen wurde.

Made available in DSpace on 2014-12-17T15:34:44Z (GMT). No. of bitstreams: 1 RenataMAC_DISSERT.pdf: 1125273 bytes, checksum: 6bd33833f33ca21674933995a1ccf14a (MD5) Previous issue date: 2012-02-28 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The aim of this study was to evaluate and characterize phenotypically goats with different levels of resistance to gastrointestinal nematodes. For a period of 93 days, 60 F2 goats originated from ? Saanen and ? Anglo- nubian animals were kept in the same area of pasture. Every seven days, feces and blood were collected for eggs per gram counts of feces (EPG) and cultures of feces and to determinate the number of eosinophils, packed cell volume and total plasma protein, respectively. On the same day, the animals were weighed and submitted to body score condition and FAMACHA method to worm control. Based on the average of EPG, the twelve animals with the highest average (susceptible group) and the twelve animals with the lowest average of EPG (resistant group) were selected, slaughtered and necropsied to recovery, counting andparasites identification. The resistant animals present lower EPG mean (P <0.0001) and 4.7 folder less parasites than susceptible animals. The resistant group presented higher mean packed cell volume (26.48%) and total plasma protein (6.24 g / dl) than susceptible one (24,04% e 5,82g/dl, respectively). The average number of eosinophils was similar in both groups The Haemonchus sp. was the most prevalent in the culture of feces, followed by Trischostrongylus sp. and Oesophagostomum sp.. The counting of nematodes in the abomasum of susceptible group was higher than in resistant one. The species identified were H. contortus in abomasums and T. colubriformis in small intestine. It can be concluded that EPG, packed cell volume and total plasma protein were useful phenotypic markers to identify animals as resistant and susceptible to gastrointestinal nematodes infections / O presente trabalho teve como objetivo de avaliar e caracterizar fenotipicamente caprinos com diferentes n?veis de resist?ncia a nematoides gastrintestinais. Em um per?odo de 93 dias, 60 caprinos F2 oriundos do cruzamento de animais ? Saanen e ? Anglo-nubiano foram mantidos em uma mesma ?rea de pastagem cultivada irrigada de capim Tanz?nia (Panicum maximum Jacq. Cv Tanz?nia). A cada sete dias, fezes e sangue foram coletados para contagem de ovos por grama de fezes (OPG), coproculturas, contagem de eosin?filos, determina??o de volume globular e prote?na plasm?tica total, respectivamente. No mesmo dia das coletas, os animais foram pesados e avaliados quanto ao escore da condi??o corporal e grau de anemia com aux?lio no cart?o FAMACHA. Com base na m?dia de OPG, os doze animais com as maiores m?dias de OPG (grupo suscept?vel) e os doze animais com as menores m?dias de OPG (grupo resistente) foram identificados e selecionados para serem abatidos e necropsiados para a recupera??o, contagem e identifica??o dos parasitos presentes. Os animais pertencentes ao grupo resistente apresentaram menor m?dia de OPG (P<0,0001) e 4,7 vezes menos parasitos adultos do que os animais do grupo suscept?vel. O grupo resistente obteve maior m?dia de volume globular (26,48%) e prote?na plasm?tica total (6,24 g/dl) do que os animais suscept?veis (24,04% e 5,82g/dl, respectivamente). A m?dia de eosin?filos foi semelhante nos dois grupos .O g?nero Haemonchus foi o mais prevalente a nas coproculturas, seguido por Trischostrongylus e Oesophagostomum. A contagem de nematoides foi maior no abomaso do grupo suscept?vel do que no grupo resistente. As esp?cies identificadas foram H. contortus no abomaso e T. colubriformis no intestino delgado. Conclui-se que OPG, volume globular e prote?na plasm?tica total foram marcadores fenot?picos eficientes para identificar animais resistentes e suscept?veis ?s infec??es causadas por nematoides gastrintestinais

No description available.

Proteins, which regulate most of the biological activities, perform their functions through their unique three-dimensional structures. The folding process of this three dimensional structure from one dimensional sequence is not well understood. The available facts infer that the protein structures are mostly conserved while sequences are more tolerant to mutations i.e. a number of sequences can adopt the same fold. These arch of optimal sequences for a chosen conformation is known as inverse protein folding and this thesis takes this approach to solve the enigmatic problem. This thesis presents a protein sequence design method based on the native state topology of protein structure. The structural importance of the amino acid positions has been converted into the topological parameter of the protein conformation. This scheme of extraction of topology of structures has been successfully applied on three dimensional lattice structures and in turn sequences with minimum energy for a given structure are obtained. This technique along with the reduced amino cid alphabet(A reduced amino acid alphabet is any clustering of twenty amino acids based on some measure of the irrelative similarity) has been applied on the protein structures and hence designed optimal amino acid sequences for a given structure. These designed sequences are energetically much better than the native amino acid sequence. The utility of this method is further confirmed by showing the similarity between naturally occurring and the designed sequences. In summary, a computationally efficient method of designing optimal sequences for a given structure is given. The physical interaction energy between the amino acids is an important part of study of protein-protein interaction, structure prediction, modeling and docking etc. The local environment of amino acids makes a difference between the same amino acid pairs in the protein structure and so the pair-wise interaction energy of amino acid residues should depend on the irrespective environment. A local environment depended knowledge based potential energy function is developed in this thesis. Two different environments, one of these is the local degree (number of contacts) and the other is the secondary structural element of amino acids, have been considered. The investigations have shown that the environment-based interaction preferences for amino acids is able to provide good potential energy functions which perform exceedingly well in discriminating the native structure from the structures with random interactions. Further, the membrane proteins are located in a completely different physico-chemical environment with different amino acid composition than the water soluble proteins. This work provides reliable potential energy functions which take care of different environment for the investigation(model/predict) of the structure of helical membrane proteins. Three different environments, parallel and perpendicular to the lipid bilayer and number of amino acid contacts, are explored to analyze the environmental effects on the potential functions. These environment dependent scoring functions perform exceedingly well indiscriminating the native sequence from a set of random sequences. Hydrophobicity of amino acids is a measure of buriedness or exposure to the aqueous environment. The lack of uniformity within the protein environment gives rise to the different values of hydrophobicity for the same amino acids, which completely depends on its location inside the protein.The contact based environment dependent hydrophobicity values of all amino acids, separately for globular and membrane proteins, have also been evaluated in this thesis. Apart from developing scoring functions, the packing of helices in membrane proteins is investigated by an approach based on the local backbone geometry and side chain atom-atom contacts of amino acids. A parameter defined in this study is able to capture the essential features of inter-helical packing, which may prove to be useful in modeling of helical membrane proteins. In conclusion, this thesis has described a novel technique to design the energetically minimized amino acid sequences which can fold in to a given conformation. Also the environment dependent interaction preference of amino acids in globular proteins is captured an efficient manner. Specially, the environment dependent scoring function for helical membrane proteins is a first successful attempt in this direction.

Membrane proteins roughly constitute 30% of open reading frames in a genome and form 70% of current drug targets. They are classified as integral, peripheral membrane proteins and polypeptide toxins. α-helices and β -strands are the principal secondary structures observed in integral membrane proteins. This thesis presents the results of studies on analysis and correlation of sequence and structure of helices constituting integral helical membrane proteins. The aim of this work is to understand the helix stabilization, distortion as well as packing in terms of amino acid sequences and the correlated structures they adopt. To this end, analyses of datasets of X-ray crystal structures of integral helical membrane proteins and their comparison with a dataset of representative folds of globular proteins was carried out. Initial analysis was carried out using a non-redundant dataset of 75 membrane proteins to understand sequence and structural preferences for stabilization of helix termini. The subsequent analysis of helix distortions in membrane proteins was carried out using an updated dataset of 90 membrane proteins. Chapter 1 of the thesis reviews experimental as well as theoretical studies that have provided insights into understanding the structure of helical membrane proteins. Chapter 2 details the methods used during the course of the present investigations. These include the protocol used for creation of the non-redundant database of membrane and globular proteins. Various statistical methods used to test significance of the position-wise representation of amino acids in helical regions and the differences in globular and membrane protein datasets have been listed. Based on the tests of significance, a methodology to identify differences in propensity values that are statistically significant among two datasets has been devised. Programs used for secondary structure identification of membrane proteins namely Structure Identification (STRIDE) and Assignment of Secondary Structure in Proteins (ASSP) as well as those used for characterization of helical geometry (Helanal-Plus) have also been enlisted. In Chapter 3, datasets of 865 α-helices in 75 membrane proteins and 2680 α- helices from 626 representative folds in globular proteins defined by the STRIDE program have been analyzed to study the sequence determinants at fifteen positions within and around the α-helix. The amino acid propensities have been studied for positions that are important for the process of helix initiation, propagation, stabilization and termination. Each of the 15 positions has unique sequence characteristics reflecting their role and contribution towards the stability of the α-helix. A comparison of the sequence preferences in membrane and globular proteins revealed common residue preferences in both these datasets confirming the importance of these positions and the strict residue preferences therein. However, short/medium length α-helices that initiated/terminated within the membrane showed distinct amino acid preferences at the N-terminus (Ncap, N1, N2) as well as the C-terminus ( Ccap, Ct) when compared to α-helices belonging to membrane and globular proteins. The sequence preferences in membrane proteins were governed by the helix initiating and terminating property of the amino acids as well as the external environment of the helix. Results from our analysis also conformed well with experimentally tested amino acid preferences in a position-specific amino acid preference library of the rat neurotensin receptor (Schlinkmann et al (2012) Proc Natl Acad Sci USA 109(25):1890-5) as well as crystal structures of GPCR proteins. In the light of the environment dependent amino acid preferences found at α- helix termini, a survey was carried out to find various helix capping motifs adopted at both termini of α-helices in globular and membrane proteins to stabilize these helix termini. The results from these findings have been reported in Chapter 4. A sequence dependent structural preference is found for capping motifs at helix termini embedded inside and protruding outside the membrane. The N-terminus of α-helices was capped by hydrogen bonds involving free main chain amide groups of the first helical turn as donors and amino acid side chains as acceptors, as against the C-terminus which showed position-dependent characteristic backbone conformations to cap the helix. Overall helix termini inside the membrane did not show a very high number of capping motifs; instead these termini were stabilized by helix- helix interactions contributed by the neighboring helices of the helical bundle. In Chapter 5, we examine transmembrane helical (TMH) regions to identify as well as characterize the various types of helix perturbations in membrane proteins using ASSP and Helanal-Plus. A survey of literature shows that the term ‘helix kink’ has been used rather loosely when in fact helical regions show significant amounts of variation and transitions in helical parameters. Hence a systematic analysis of TMH regions was undertaken to quantify different types of helix perturbations, based on geometric parameters such as helical twist, rise per residue and local bending angle. Results from this analysis indicated that helices are not only kinked but undergo transitions to form interspersed stretches of 310 helices and π-bulges within the bilayer. These interspersed 310 and π-helices showed unique sequence preferences within and around their helical body, and also assisted in main- taining the helical structure within the bilayer. We found that Proline not only kinked the helical regions in a characteristic manner but also caused a tightening or unwinding in a helical region to form 310 and π-helix fragments respectively. The helix distortions also resulted in backbone hydrogen bonds to be missed which were stabilized by hydrogen bonds from neighboring residues mediated by their side chain atoms. Furthermore, a packing analysis showed that helical regions with distortions were able to establish inter-helical interactions with more number of transmembrane segments in the helical bundle. The study on helix perturbations presented in the previous chapter, brought to light a previously unreported 19 amino acid π-helix fragment interspersed between α-helices in the functionally important transmembrane helix 2 (TM2) belonging to Mitochondrial cytochrome-c-oxidase (1v55). Chapter 6 describes a case study of the structurally similar but functionally different members within the Heme-Copper- Superoxidases (HCO) superfamily that were considered for a comparative analysis of TM2. An analysis of 7 family members revealed that the π-helix shortens, fragments in two shorter π-helices or was even absent in some family members. The long π-helix significantly decreased the total twist and rise of the entire helical fragment thus accommodating more hydrophobic amino acids within the bilayer to avoid hydrophobic mismatch with the bilayer. The increased radius of the TM2 helical fragment also assisted in helix packing interactions by increasing the number of residues involved in helix-helix interactions and hydrogen bonds. Chapter 7 documents the conclusions from the different analyses presented in each of the above chapters. Overall, it is found that membrane proteins optimize the biophysical and chemical constraints of the external environment to strategically place select amino acids at helix termini to ‘start’ and ‘stop’ α-helices. The stabilization of these helix termini is a consequence of sequence dependent structural preferences to form helix capping motifs. The studies on helix transitions and distortions highlight that membrane proteins are not only packed as α-helices but also accomodate 310- and π-helical fragments. These transitions and distortions help in harboring more hydrophobic amino acids and aiding inter-helical interactions important for maintaining the fold of the membrane protein. Appendix A describes a comparison of α-helix assignments in globular and membrane proteins by two algorithms, one based on Cα trace (ASSP) and the other using a combination of hydrogen bond pattern along with backbone torsion angles φ and ψ (STRIDE).

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In the standard model of cosmology (ΛCDM) the first stars, star clusters, and galaxies are expected to have formed in short bursts of star formation in low-mass dark matter halos at high redshifts (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?z%5C,%20%5Csim%20%5C,6-10" data-classname="equation_inline" />). Up to this point, attempts to predict the properties and abundances of these luminous objects have made use of numerically expensive cosmological simulations. On top of being numerically expensive, these simulations often lack the required sub-parsec resolution needed to resolve the formation of compact star clusters and/or neglect possibly dominant stellar feedback processes. Motivated by this, I introduce Anaxagoras, as far as I know the most detailed analytical ab initio model of starbursts in low-mass halos to date. The model incorporates sub-models for gas cooling (including a new determination of the H2-cooling threshold in minihalos), central gas accretion and disk formation (using a new selfsimilarsolution), stellar feedback from radiation pressure (direct stellar radiation, Lyman-<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Calpha" data-classname="equation_inline" data-title="" /> scattering in H I, and multiple scattering of IR photons by dust), stellar winds, expanding HII regions, and (crudely) supernovae. The resulting star formation efficiency is used to predict the fraction of stars that remain gravitationally bound in a cluster following gas expulsion, andwhat fraction escape the central region of the halo, yet remain bound by the dark matter halo. I apply Anaxagoras to study star formation at <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?z%5C,%20%3E%20%5C,6" data-classname="equation_inline" /> in satellite halos of the Milky Way using a halo merger tree code, as well as Population III (Pop III) star formation in minihalos. For the Milky Way setup, hundreds of galaxies are predicted to form with luminosities (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?L_%7B%5Crm%20V%7D%20%5C,%3C%5C,%20%5Crm%7Bfew%7D%5C,%20%5Ctimes%20%5C,%2010%5E4%20%5C:%20%5Crm%7BL%7D_%7B%5Codot%7D" data-classname="equation_inline" />), half-mass radii (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Csim%2010-200%5C:%5Crm%20pc" data-classname="equation_inline" />), mass-to-light ratios (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?M/L_%7B%5Crm%20V%7D%20%5Csim%20100%20-%20%5Crm%7Bfew%7D%20%5C,%5Ctimes%5C,%2010%5E3%20%5C:%5Crm%7BM%7D_%7B%5Codot%7D/%5Crm%7BL%7D_%7B%5Codot%7D" data-classname="equation" />), and ages (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?13.18%5E%7B+0.29%7D_%7B-0.31%7D%5C:%5Crm%20Gyrs" data-classname="equation_inline" data-title="" />) in good agreement with the observed local population of Ultra-Faint Dwarfs. This shows that ΛCDM is able to explain the properties ofthe faintest dwarf galaxies without fine-tuning. Furthermore, at least ~ 40 compact (initial half-mass radii <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?%5Csim%200.1-5%5C;%5Crm%7Bpc%7D" data-classname="equation_inline" />), old (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?13.27%5E%7B+0.21%7D_%7B-0.39%7D%5C:%5Crm%20Gyrs" data-classname="equation_inline" />) globular cluster (GC) candidates with initial stellar masses <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?10%5E5%20-%2010%5E6%20%5C:%20%5Crm%7BM%7D_%7B%5Codot%7D" data-classname="equation_inline" />  are predicted to form at the center of low-mass halos, and could survive to the present-day and explain at least a fraction of the observed metal-poor GCs. Their properties are consistent with recent candidates for GCs residing in dark matter halos. Thus, Anaxagoras lends support to the viability of the scenario of GC formation in minihalos. Finally, the formation of Population III (Pop III) stars in minihalos is studied, with the conclusion that if Pop III stars are not overly massive (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?25%5C:%5Crm%7BM%7D_%7B%5Codot%7D" data-classname="equation_inline" />) between ~ 1 − 30 stars could form per minihalo at <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?z%5C,%20%3E%20%5C,20" data-classname="equation_inline" />, with the number increasing to ~ 10 − 500 stars per minihalo at <img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?z%5C,%3C%5C,15" data-classname="equation_inline" /> as Lyman-Werner feedback delay star formation until halos reach larger masses. In the case where Pop III stars are more massive (<img src="http://www.diva-portal.org/cgi-bin/mimetex.cgi?140%5C:%5Crm%7BM%7D_%7B%5Codot%7D" data-classname="equation_inline" data-title="" />) most minihalos form just a single star. Due to self-shielding of H2 in minihalos, I find that the cosmological Lyman-Werner background is insufficient to produce Pop III galaxies in atomic-cooling halos, with the implication that the number of massive Pop III galaxies/star clusters in the early Universe has been greatly overestimated in the literature that ignores self-shielding.

<p> The globular cluster system of a nearby giant elliptical galaxy, NGC 5128 is studied to place constraints on the formation history of the galaxy. In this thesis, we have identified 190 new globular clusters via radial velocity measurements, bringing the total known population of globular clusters to 605 within this galaxy. We have examined the colour and spatial distributions of the globular cluster system and find it is bimodal in colour, with both a red and blue globular cluster population. The blue population is more spatially extended than the red, and both populations fall off in number density with radius as a power-law. There is a clear lack of globular clusters along the isophotal minor axis of the galaxy beyond a galactocentric radius of 15' warranting further search. With this new dataset, we have measured the ages, metallicities, and formation timescales for 72 globular clusters. The spectroscopic metallicity distribution function is bimodal indicating there is a metal-rich and metal-poor globular cluster population that corresponds to the red and blue globular clusters, respectively. We find the majority of both metal-rich (56%) and metal-poor (92%) globular clusters are older than 8 Gyr, comparable to the Milky Way globular cluster system. We do find a smaller fraction, 18% of our sample, are metal-rich globular clusters with ages younger than 5 Gyr, while the remaining globular clusters have intermediate ages between 5 -8 Gyr. The formation times of these globular clusters, estimated by their alpha-to-iron abundance ratios, indicate they formed quickly, on a timescale similar to globular clusters in most spiral galaxies, but on slower timescales than those in some other giant elliptical galaxies. The kinematics of the full globular cluster system is analyzed, as well as for the metal-rich and metal-poor globular clusters separately, as a function of galactocentric radius. We find the metal-poor globular cluster system has a small rotation signature of 17±14 km s-1 around no clearly defined axis and its dynamics are dominated by dispersion. The metal-rich globular cluster system has a mild rotation of 41 ± 15 km s-1 about the galaxy's isophotal major axis, following the rotation of a representative field star population, the planetary nebulae. The motion of the metal-rich globular cluster system is also dominated by random motion. We estimate the mass of the galaxy to be (5.5 ± 1.9) x 1011 with a mass-to-light ratio of 15.35 M0 /L80 using the globular cluster M0 population out to 20'. This estimate places NGC 5128 on a mass scale similar to other giant elliptical galaxies. The evidence collected suggests that NGC 5128 formed in a hierarchical scenario, gradually building up larger structure from smaller protogalaxies at early times in the history of the Universe. The group environment of NGC 5128 may have prolonged star formation within the galaxy as well, enabling a small spread in the old ages of globular clusters and also slowing the formation timescales compared to globular clusters in other giant elliptical galaxies. Results from this thesis also support more recent accretions in the history of NGC 5128, building up the more metal-rich and young globular clusters, which have a different rotation axis than the rest of the population. </p> / Thesis / Doctor of Philosophy (PhD)