Selective pressures at the DNA level shape genes into pro les consisting of patterns of rapidly evolving sites and sites withstanding change. These pro les remain detectable even when protein sequences become extensively diverged. It has been hypothesised that these patterns can be used as gene identi ers. A common task in molecular biology is to infer functional, structural or evolutionary relationships by querying a database using an algorithm. However, problems arise when sequence similarity is low. The problem is that the algorithm produces numerous false positives when highly conserved datasets are aligned. To increase the sensitivity of the algorithm, the evolutionary rate based approach was reimplemented and coupled with a conventional BLOSUM substitution matrix to produce a new implementation called BLOSUM-FIRE. The two approaches are combined in a dynamic scoring function, which uses the selective pressure to score aligned residues. Analysis of quality of alignments produced, revealed that the new implementation of the FIRE algorithm performs as well as conventional algorithms. In addition, the Evolutionary rate Database (EvoDB), which is a compilation of evolutionary rate pro les of all the members of the PFAM-A protein domain database has been developed. The EvoDB database can be queried using FIRE to infer protein domain functions. The utility of this algorithm and database was tested by inferring the domain functions of the Hepatitis B X protein. Results show that the BLOSUM-FIRE algorithm was able to accurately identify the domain function of HBx as a trans-activation protein using EvoDB. The biological relevance of these results was not validated and requires further interrogation; however, these proteins share vital roles in viral replication. This study demonstrates the utility of an evolutionary rate based approach and demonstrates that such an approach is robust when coupled with an amino acid substitution matrix yielding results comparable to conventional algorithms. EvoDB is a catalogue of the evolutionary rate pro les and provides the corresponding phylogenetic trees, PFAM-A alignments and annotated accession identi er data. The BLOSUM-FIRE software and user manual including the EvoDB at le database and release notes have been made freely available at www.bioinf.wits.ac.za/software/fire. The BLOSUM-FIRE algorithm and EvoDB database present a tier of information untapped by current databases and tools. / A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in ful lment of the requirements of the degree of Master of Science (Medicine).

To establish comprehensive pharmacology of P2X₇ receptors, membrane current recording, intracellular calcium transient recording and ethidium bromide uptake were carried out to examine several selective (A-740003, A- 438079) and non-selective (suramin) P2X₇ antagonists across mammalian P2X₇ receptors (human, mouse and rat). These P2X₇ receptors demonstrated species-dependent sensitivities to antagonists. In each species, A-740003 revealed variant IC50 values with different assays, indicating the assay- dependent pharmacology of P2X₇ receptors. Conventionally, pharmacology can be used to define a native current but not in the case of the human breast cancer cell line, Hs578T. It is found that P2X₇ was expressed at both mRNA and protein level. The ATP-evoked currents recorded from Hs578T cells were P2X₇-like with distinctive electrophysiological features. But the pharmacology profile of the currents did not fit with P2X₇ receptor. Further experiments are needed to either include or exclude the existence of functional P2X₇ receptors in Hs578T. Transmembrane domain 2 (TM2) is known as the pore-forming region for P2X receptors. TM2 of P2X₇ receptor was investigated with cysteine substitution scanning. The predicted α-helix structure of the TM2 segment was in good agreement with the results from the substituted cysteine accessibility method (SCAM). Thr336, Ser339, Tyr343, Phe344 and Thr348 were found important for both channel dilation and aqueous pore formation. Ser339 was further studied. Various substitutions at Ser339 were explored. The results suggest that the polarity of the side chain at Ser339 is essential for the channel dilation. Furthermore, disulfide bond formation was identified between S339C in the trimeric receptor, implying that the side chains of Ser339 might turn very close to each other during the channel opening and dilation.

Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein's interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may "fine-tune" the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates. / Ph. D.

The FeMo cofactor is one of two types of prosthetic group found in the larger of the two nitrogenase component proteins, called the MoFe protein, and it is strongly implicated as the substrate binding and reduction site. The glutamine-191 residue in the Alpha-subunit of the MoFe protein of A. vinelandii nitrogenase was targeted for substitution because its side chain is involved in a hydrogen-bond network from one of the terminal carboxylates of the homocitrate component of FeMo cofactor through to the backbone NH of Alpha Gly-61, which is adjacent to Alpha Cys-62, which ligates to the P cluster (the second type of prosthetic group in the MoFe protein). A variety of altered MoFe proteins produced by the A. vinelandii mutant strains, namely the Alpha Pro-191, Alpha Ser-191, Alpha Thr-191, Alpha His-191, Alpha Glu-191, and Alpha Arg-191 altered MoFe proteins, have been purified to homogeneity and the catalytic properties of these altered MoFe proteins have been compared to those of wild type MoFe protein. Unlike wild type, the six altered MoFe proteins have decreased catalytic activity on substrate reduction and exhibited H2 evolution that was partially inhibited by added CO. Moreover, some of altered MoFe proteins with lower specific activity for the C2H4 production can produce C2H6 from C2H2. The results from the pH and activity studies indicate that the substitutions on the MoFe protein have an effect on the contribution of the responsible acid-base group(s) involved in proton transfer for H+- and C2H2-reduction. Furthermore, the inhibition by CO of hydrogen evolution by these altered MoFe proteins is likely from a lowering of the rate of both electron and proton transfer to the H+- reduction site(s). Some altered MoFe proteins but not wild type MoFe protein can produce C2H6 from C2H2. This observation suggested a lower apparent binding affinity for C2H2 and a slower proton transfer to C2H2 reduction with these altered MoFe proteins, which allow the intermediate to stay at the site longer and be further reduced by two electrons and two protons to give C2H6. These changes in the biochemical properties of these altered MoFe proteins indicate that the Alpha Gln-191 residue is intimately involved in substrate binding and reduction including proton delivery to substrate. / Ph. D.

The advent of high-throughput sequencing and structure determination techniques has had a tremendous impact on our quest in cracking the language of life. The genomic and protein data is now being accumulated at a phenomenal rate, with the motivation of deriving insights into the function, mechanism, and evolution of the biomolecules, through analysis of their similarities, differences, and interactions. The rapid increase in the size of the biomolecular databases, however, calls for development of new computational methods for sensitive and efficient management and analysis of this information. In this thesis, we propose and implement several approaches for accurate and highly efficient comparison and retrieval of protein sequences and structures. The observation that corresponding residues in related proteins share similar inter-residue contacts is exploited in derivation of a new set of biologically sensitive metric amino acid substitution matrices, yielding accurate alignment and comparison of proteins. The metricity of these matrices has allowed efficient indexing and retrieval of both protein sequences and structures. A landmark-guided embedding of protein sequences is developed to represent subsequences in a vector space for approximate, but extremely fast spatial indexing and similarity search. Whereas protein structure comparison and search tasks were hitherto handled separately, we propose an integrated approach that serves both of these tasks and performs comparable to or better than other available methods. Our approach hinges on identification of similar residue contacts using distance-based indexing and provides the best of the both worlds: the accuracy of detailed structure alignment algorithms, at a speed comparable to that of the structure retrieval algorithms. We expect that the methods and tools developed in this study will find use in a wide range of application areas including annotation of new proteins, discovery of functional motifs, discerning evolutionary relationships among genes and species, and drug design and targeting.

Thesis (Ph.D. in Neuroscience) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 108-118). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

This thesis discusses the issue of predicting of the effect of amino acid substitutions on protein funkcion, based on phylogenetic analysis method, inspired by tool MAPP. Significant number of genetic diseases is caused by nonsynonymous SNPs manifested as single point mutations on the protein level. The ability to identify deleterious substitutions could be useful for protein engineering to test whether the proposed mutations do not damage protein function same as for targeting disease causing harmful mutations. However the experimental validation is costly and the need of predictive computation methods has risen. This thesis describes desing and implementation of a new in silico predictor based on the principles of evolutionary analysis and dissimilarity between original and substituting amino acid physico-chemical properties. Developed algorithm was tested on four datasets with 74,192 mutations from 16,256 sequences in total. The predictor yields up to 72 % accuracy and in the comparison with the most existing tools, it is substantially less time consuming. In order to achieve the highest possible efficiency, the optimization process was focused on selection of the most suitable (a) third-party software for calculation of a multiple sequence alignment, (b) overall decision threshold and (c) a set of physico-chemical properties.

Flavonoids are secondary metabolites that are important in plant defense, protection and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. A flavonol-specific 3-O-GT enzyme has been identified and cloned from leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3-O-GTs from grape (Vitis vinifera) and grapefruit (Citrus paradisi) were modeled against F7-O-GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-O-GT that were identified through homology modeling. Analyses of the enzyme activity of the mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzyme inactive with flavonol substrates. Mutation of glycine 392 to glutamate (G392E) was active at 80% relative to the wild type. The mutant enzyme also did not show broadened acceptor specificity as it also favored flavonols as the preferred acceptor substrate. The glucosylation products of the active mutant enzyme will be analyzed to determine if this resulted in a change in regiospecificity.

Meiosis is functionally conserved across eukaryotes, thus not expected to vary considerably among different species, and even less so among lineages within a species. However, recent studies showed that this is not necessarily the case in Arabidopsis arenosa. Genome scanning identified an excess differentiation in meiosis genes between A. arenosa diploids and tetraploids, interpreted as meiosis adaptation to the whole genome duplication in tetraploids and differentiation was also found between two diploid lineages. Thus, I present a population-based analysis of positive selection acting on meiosis proteins across multiple lineages of A. arenosa. I showed that meiosis proteins were under positive selection in all diploid lineages, mainly in the Pannonian and South-eastern Carpathian lineage. The evidence for positive selection in diploid lineages suggested differential pathways of meiosis adaptations in the species, probably reflecting the necessity to adapt to local environments, among all to temperature. The highest enrichment of amino acid substitutions (AASs) under positive selection was identified in tetraploids, in consistence with previous genome-scan results. As several interacting meiosis proteins were under positive selection in the same A. arenosa lineage, I hypothesize that the close...

This thesis discusses the issue of predicting the effect of amino acid substitutions on protein function using MAPP method. This method requires the multiple sequence alignment and phylogenetic tree constructed by third-party tools. Main goal of this thesis is to find the combination of suitable tools and their parameters to generate the inputs of MAPP method on the basis of analysis on one massively mutated protein. Then, the MAPP method is tested with chosen combination of parameters and tools on two large independent datasets and consequently is compared with the other tools focused on prediction of the effect of mutations. Apart from this the web interface for the MAPP method was created. This interface simplifies the use of the method since the user need not to install any tools or set any parameters.