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1	Structural and Functional Aspects of Evolutionarily Conserved Signature Indels in Protein Sequences. Khadka, Bijendra January 2019 (has links) Analysis of genome sequences is enabling identification of numerous novel characteristics that provide valuable means for genetic and biochemical studies. Of these characteristics, Conserved Signature Indels (CSIs) in proteins which are specific for a given group of organisms have proven particularly useful for evolutionary and biochemical studies. My research work focused on using comparative genomics techniques to identify a large number of CSIs which are distinctive characteristics of fungi and other important groups of organisms. These CSIs were utilized to understand the evolutionary relationships among different proteins (species), and also regarding their structural features and functional significance. Based on multiple CSIs that I have identified for the PIP4K/PIP5K family of proteins, different isozymes of these proteins and also their subfamilies can now be reliably distinguished in molecular terms. Further, the species distribution of CSIs in the PIP4K/PIP5K proteins and phylogenetic analyses of these protein sequences, my work provides important insights into the evolutionary history of this protein family. The functional significance of one of the CSI in the PIP5K proteins, specific for the Saccharomycetaceae family of fungi, was also investigated. The results from structural analysis and molecular dynamics (MD) simulation studies show that this 8 aa CSI plays an important role in facilitating the binding of fungal PIP5K protein to the membrane surface. In other work, we identified multiple highly-specific CSIs in the phosphoketolase (PK) proteins, which clearly distinguish the bifunctional form of PK found in bifidobacteria from its homologs (monofunctional) found in other organisms. Structural analyses and docking studies with these proteins indicate that the CSIs in bifidobacterial PK, which are located on the subunit interface, play a role in the formation/stabilization of the protein dimer. We have also identified 2 large CSIs in SecA proteins that are uniquely found in thermophilic species from two different phyla of bacteria. Detailed bioinformatics analyses on one of these CSIs show that a number of residues from this CSI, through their interaction with a conserved network of water molecules, play a role in stabilizing the binding of ADP/ATP to the SecA protein at high temperature. My work also involved developing an integrated software pipeline for homology modeling of proteins and analyzing the location of CSIs in protein structures. Overall, my thesis work establishes the usefulness of CSIs in protein sequences as valuable means for genetic, biochemical, structural and evolutionary studies. / Dissertation / Doctor of Philosophy (PhD)
2	Análise da hidrofobicidade na evolução de proteínas Silva, Ricardo Hildebrand Theodoro da [UNESP] 28 September 2009 (has links) (PDF) Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-28Bitstream added on 2014-06-13T19:19:35Z : No. of bitstreams: 1 silva_rht_dr_sjrp.pdf: 929264 bytes, checksum: 5ac0a998512f2430142616c64c6de249 (MD5) / Efeito das mutações sobre a estabilidade das proteínas e uma questão crucial na evolução da proteína. Tais efeitos dependem fortemente do car ater hidrofóbico global da proteína. Em um trabalho recente (J. Chem. Phys.125,084904(2006), n os sugerimos dois cenários de enovelamento com consequências distintas ma evolução da proteína. O limite de baixa hidrofobicidade, corresponde ao regime em que ocorre concomitantemente o colapso e a formação da estrutura nativa. Sob estas condições as proteínas são pouco robustas a mutações, o que implica em uma alta homologia entre proteínas de diferentes espécies. O limite de alta hidrofobicidade, corresponde ao regime em que a proteína sofrem um colapso antes do enovelamento, e neste caso as proteínas são mais robustas a proteínas, sugerindo uma menor homologia entre proteínas de diferentes espécies. Neste trabalho, n os estudamos a homologia de quatro proteínas para 41 espécies diferentes, correlacionando as suas homologias com suas hidrofobicidades médias. As proteínas estudadas foram lisozima, citocromo-c, mioglobina e histona H3, utilizando seis escalas hidrofóbicas diferentes. Junto com o cálculo da homologia, foi realizada uma comparação da similaridade estrutural (rmsd). Os resultados con rmam a hipótese acima, indicando que proteínas, em condições de baixa hidrofobicidade, têm baixa variabilidade de sequências e conformações, para alta hidrofobicidade, as proteínas exibem variabilidade de sequências e conformações / Efect of mutations on stability of proteins is a crucial issue in protein evolution. Such e ects depend strongly on the overall hydrophobic protein character. In a recent work we suggested two scenarios for folding with distinct protein evolution consequences (J. Chem. Phys.125 084904,2006) Under low hydrophobic conditions proteins collapse concomitantly with the formation of their native state, and are less robust to mutations, which implies higher homology among proteins of di erent species. On the other limit, at high hydrophobicity proteins collapse before folding, and in this case they are more susceptible to mutations, suggesting lower homology among proteins of di erent species. In this work we investigate this conjecture studying the homology of four proteins for 41 di erent species, correlating it with their average hydrophobicity. The proteins studied were lysozyme, cytochrome-c, myoglobin and histone H3, using six di erent hydrophobic scales. Along with the homology calculation, a comparison of structural similarity (rmsd) was also carried out. The results con rm the above hypothesis, indicating that proteins at low hydrophobicity display low variations on sequences and conformations. On other hand, at high hydrophobicity, proteins exhibit high variability on sequences and conformations. Keywords: evolution, protein folding, scenarios of folding, projetabilidade, hydrophobicity, homology of proteins, mutations in proteins Biofísica Biologia molecular Proteínas Biofísica molecular Enovelamento de proteínas Evolução de proteínas Protein evolution Scenarios of foldin Protein folding Hydrophobicity Homology of protein Mutations in proteins
3	Análise da hidrofobicidade na evolução de proteínas / Silva, Ricardo Hildebrand Theodoro da. January 2009 (has links) Resumo: Efeito das mutações sobre a estabilidade das proteínas e uma questão crucial na evolução da proteína. Tais efeitos dependem fortemente do car ater hidrofóbico global da proteína. Em um trabalho recente (J. Chem. Phys.125,084904(2006), n os sugerimos dois cenários de enovelamento com consequências distintas ma evolução da proteína. O limite de baixa hidrofobicidade, corresponde ao regime em que ocorre concomitantemente o colapso e a formação da estrutura nativa. Sob estas condições as proteínas são pouco robustas a mutações, o que implica em uma alta homologia entre proteínas de diferentes espécies. O limite de alta hidrofobicidade, corresponde ao regime em que a proteína sofrem um colapso antes do enovelamento, e neste caso as proteínas são mais robustas a proteínas, sugerindo uma menor homologia entre proteínas de diferentes espécies. Neste trabalho, n os estudamos a homologia de quatro proteínas para 41 espécies diferentes, correlacionando as suas homologias com suas hidrofobicidades médias. As proteínas estudadas foram lisozima, citocromo-c, mioglobina e histona H3, utilizando seis escalas hidrofóbicas diferentes. Junto com o cálculo da homologia, foi realizada uma comparação da similaridade estrutural (rmsd). Os resultados con rmam a hipótese acima, indicando que proteínas, em condições de baixa hidrofobicidade, têm baixa variabilidade de sequências e conformações, para alta hidrofobicidade, as proteínas exibem variabilidade de sequências e conformações / Abstract: Efect of mutations on stability of proteins is a crucial issue in protein evolution. Such e ects depend strongly on the overall hydrophobic protein character. In a recent work we suggested two scenarios for folding with distinct protein evolution consequences (J. Chem. Phys.125 084904,2006) Under low hydrophobic conditions proteins collapse concomitantly with the formation of their native state, and are less robust to mutations, which implies higher homology among proteins of di erent species. On the other limit, at high hydrophobicity proteins collapse before folding, and in this case they are more susceptible to mutations, suggesting lower homology among proteins of di erent species. In this work we investigate this conjecture studying the homology of four proteins for 41 di erent species, correlating it with their average hydrophobicity. The proteins studied were lysozyme, cytochrome-c, myoglobin and histone H3, using six di erent hydrophobic scales. Along with the homology calculation, a comparison of structural similarity (rmsd) was also carried out. The results con rm the above hypothesis, indicating that proteins at low hydrophobicity display low variations on sequences and conformations. On other hand, at high hydrophobicity, proteins exhibit high variability on sequences and conformations. Keywords: evolution, protein folding, scenarios of folding, projetabilidade, hydrophobicity, homology of proteins, mutations in proteins / Orientador: Vitor Barbanti Pereira Leite / Coorientador: Jorge Chahine / Banca: Antonio Caliri / Banca: Eduardo Beleza Yamagi / Banca: Luis Paulo Barbour / Banca: Sidnei Jhourado de Carvalho / Doutor Biofísica. Biologia molecular. Proteínas. Protein evolution. eng Scenarios of foldin. eng Protein folding. eng Hydrophobicity. eng Homology of protein. eng Mutations in proteins. eng
4	The Characterisation of Putative Nuclear Pore-Anchoring Proteins in Arabidopsis thaliana Collins, Patrick January 2013 (has links) The nuclear pore complex (NPC) is perhaps the largest protein complex in the eukaryotic cell, and controls the movement of molecules across the nuclear envelope. The NPC is composed of up to 30 proteins termed nucleoporins (Nups), each grouped in different sub-complexes. The transmembrane ring sub-complex is composed of Nups responsible for anchoring the NPC to the nuclear envelope. Bioinformatic analysis has traced all major sub-complexes of the NPC back to the last eukaryotic common ancestor, meaning that the nuclear pore structure and function is conserved amongst all eukaryotes. In this study Arabidopsis T-DNA knockout lines for these genes were investigated to characterise gene function. Differences in plant growth and development were observed for the ndc1 knockout line compared to wild-type but gp210 plants showed no phenotypic differences. The double knockout line gp210 ndc1 was generated through crosses to observe plant response to the knockout of two anchoring-Nup genes. No synergistic affect from this double knockout was observed, suggesting that more, as yet unidentified Nups function the transmembrane ring in plants. The sensitivity to nuclear export inhibitor leptomycin B (LMB) was tested also for knockout lines, although growth sensitivity to the drug was not observed. Nucleocytoplasmic transport of knockout lines was measured in cells transformed by particle bombardment. To express fluorescent protein constructs actively transported through the NPC, localisation of protein determined the nucleocytoplasmic transport of the cell. The ndc1single knockout and the double knockout gp210 ndc1 exhibited decreased nuclear export. Further experiments in determining NDC1 localisation and identification of other Nups in the transmembrane ring sub-complex would bring a more comprehensive understanding to the plant NPC. Arabidopsis thaliana transformation insert transient expression epidermal cells root cells gene protein plant bolting flowering root growth knockout ndc1 gp210 At1g73240 At5g40480 SALK SAIL Columbia seed germination DNA sequencing light microscopy stereo-fluorescence microscopy DNA extraction cell nucleus cytoplasm nucleoplasm nuclear pore complex nucleoporin nuclear envelope putative nuclear pore-anchoring protein leptomycin B gene gun particle bombardment agrobacterium T-DNA fasciation rhodococcus fascians cross reverse genetics nucleocytoplasmic transport cross self synergistic silique transmembrane ring dihybrid pumilo homology domain protein GFP YFP RFP PCR homozygote heterozygote inhibition

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