In higher plants oxygenic photosynthesis primarily takes place in the chloroplasts of leaves. Within the chloroplasts is an intricate membrane system, the thylakoid membrane, which is the site of light harvesting and photosynthetic electron transport. Enclosed by this membrane is the lumen space, which initially was believed to only contain a few proteins, but now is known to house a distinct set of >50 proteins, many for which there is still no proposed function. The work presented in this thesis is focused on understanding the functions of the proteins in the lumen space. Using proteomic methods, we investigated first the regulation of lumenal proteins by light and secondly by dithiol-disulphide exchange, mediated by the disulphide reductase protein thioredoxin. We furthermore performed structural and functional studies of the lumenal pentapeptide repeat proteins and of the PsbP-domain protein PPD6. When studying the diurnal expression pattern of the lumen proteins, using difference gel electrophoresis, we observed an increased abundance of fifteen lumen protein in light-adapted Arabidopsis thaliana plants. Among these proteins were subunits of the oxygen evolving complex, plastocyanin and proteins of unknown function. In our analysis of putative lumenal targets of thioredoxin, we identified nineteen proteins, constituting more than 40 % of the lumen proteins observable by our methods. A subset of these putative target proteins were selected for further studies, including structure determination by x-ray crystallography. The crystal structure of the pentapeptide repeat protein TL15 was solved to 1.3 Å resolution and further biochemical characterization suggested that it may function as a novel type of redox regulated molecular chaperone in the lumen. PPD6, a member of the PsbP-family of proteins, which is unique in that it possesses a conserved disulphide bond not found in any other PsbP-family protein, was also expressed, purified and crystallized. A preliminary x-ray analysis suggests that PPD6 exists as a dimer in the crystalline state and binds zinc ions. The high representation of targets of thioredoxin among the lumen proteins, along with the characterization of the pentapeptide repeat protein family, implies that dithiol-disulphide exchange reactions play an important role in the thylakoid lumen of higher plants, regulating processes such as photoprotection, protein turnover and protein folding.

Background: Through the last decades vast amount of sequence information have been produced by various protein sequencing projects, which enables studies of sequential patterns. One of the bestknown efforts to chart short peptide sequences is the Prosite pattern data bank. While sequential patterns like those of Prosite have proved very useful for classifying protein families, functions etc. structural analysis may provide more information and possible crucial clues linked to protein folding. Today PDB, which is the main repository for protein structure, contains more than 50’000 entries which enables structural protein studies. Result: Strongly folded pentapeptides, defined as pentapeptides which retained a specific conformation in several significantly structurally different proteins, were studied out of PDB. Among these several groups were found. Possibly the most well defined is the “double Cys” pentapeptide group, with two amino acids in between (CXXCX|XCXXC) which were found to form backbone loops where the two Cysteine amino acids formed a possible Cys-Cys bridge. Other structural motifs were found both in helixes and in sheets like "ECSAM" and "TIKIW", respectively. Conclusion: There is much information to be extracted by structural analysis of pentapeptides and other oligopeptides. There is no doubt that some pentapeptides are more likely to obtain a specific fold than others and that there are many strongly folded pentapeptides. By combining the usage of such patterns in a protein folding model, such as the Hydrophobic-polar-model improvements in speed and accuracy can be obtained. Comparing structural conformations for important overrepresented pentapeptides can also help identify and refine both structural information data banks such as SCOP and sequential pattern data banks such as Prosite.

<p>Background: Through the last decades vast amount of sequence information have been produced by various protein sequencing projects, which enables studies of sequential patterns. One of the bestknown efforts to chart short peptide sequences is the Prosite pattern data bank. While sequential patterns like those of Prosite have proved very useful for classifying protein families, functions etc. structural analysis may provide more information and possible crucial clues linked to protein folding. Today PDB, which is the main repository for protein structure, contains more than 50’000 entries which enables structural protein studies.</p><p>Result: Strongly folded pentapeptides, defined as pentapeptides which retained a specific conformation in several significantly structurally different proteins, were studied out of PDB. Among these several groups were found. Possibly the most well defined is the “double Cys” pentapeptide group, with two amino acids in between (CXXCX|XCXXC) which were found to form backbone loops where the two Cysteine amino acids formed a possible Cys-Cys bridge. Other structural motifs were found both in helixes and in sheets like "ECSAM" and "TIKIW", respectively.</p><p>Conclusion: There is much information to be extracted by structural analysis of pentapeptides and other oligopeptides. There is no doubt that some pentapeptides are more likely to obtain a specific fold than others and that there are many strongly folded pentapeptides. By combining the usage of such patterns in a protein folding model, such as the Hydrophobic-polar-model improvements in speed and accuracy can be obtained. Comparing structural conformations for important overrepresented pentapeptides can also help identify and refine both structural information data banks such as SCOP and sequential pattern data banks such as Prosite.</p>

Protein tyrosine nitration (PTN) is a posttranslational modification resulting from oxidative/nitrosative stress that has been implicated in a wide variety of disease states. Characterization of PTN is challenging due to several factors including its low abundance in a given proteome, preferential site modification, multiple target site proximity within unique peptide sequences, and analytical method and instrument limitations. Current analytical techniques are insufficiently sensitive to identify endogenous nitration sites without incorporation of either nitrotyrosine or target protein enrichment. However, enrichment proficiency can also be inadequate. Chemical derivatization of the nitro- moiety can be incomplete or result in undesirable byproduct formation, while immunoaffinity proficiency is contingent upon antibody specificity. To overcome analytical method and enrichment deficiencies, we aimed to develop a comprehensive nitroproteome-specific workflow using molecular methods combined with mass spectrometry. Our approach was to systematically address all relevant factors contributing to PTN such as primary sequence, protein conformation, solvent accessibility, and nitrating agent concentration. Our ultimate goal was to increase mass spectrometric sensitivity for PTN identification. All putative nitroprotein/nitropeptide identifications were then subjected to rigorous validation by either manual spectrum analyses or peptide synthesis. We further developed MS methods for quantitation of nitropeptides from complex mixtures with minimal sample processing. Successful application of our nitroproteome-specific mass spectrometric workflow is expected to provide powerful tools for comprehensive PTN investigation that will elucidate its role in the onset and progression of a variety of disease states as well as facilitate discovery of therapeutic targets.

No description available.

No description available.

Sinorhizobium meliloti is a nitrogen-fixing endosymbiont of the legume Medicago sativa commonly known as alfalfa. It uses flagellar rotation and chemotaxis to seek roots of host plants to inhabit. This symbiosis serves as a great model system for studying biological nitrogen fixation and plant-microbe interactions. Since alfalfa brings enormous economic value to the USA, investments into the knowledge of the chemotaxis process that initiates symbiosis have the ability to mitigate deterioration of the environment and significantly increase food supply. The chemotaxis system in the enteric bacteria Escherichia coli is well studied and has been a great resource to understanding the process in other bacterial systems including our model organism S. meliloti. This dissertation compares and contrasts the chemotaxis features in E. coli and S. meliloti and investigates their molecular functions. Based on the understanding gained so far, we attempt to offer plausible explanations for the underlying mechanisms of the S. meliloti chemotaxis pathway. Chapter 1 describes why biological nitrogen fixation is important for agriculture and the health of our environment. This chapter also sheds light on the symbiotic relationship between alfalfa and S. meliloti, which culminates in the formation of nitrogen fixing nodules. We expound on the chemotaxis systems in E. coli and other bacteria including S. meliloti and Bacillus subtilis. In chapter 2, we compare the distribution of C-terminal pentapeptide-bearing receptors and the adaptation proteins that they tether in E. coli and S. meliloti. The stoichiometry data show that the ratio of pentapeptide-bearing chemoreceptors to chemotaxis protein (Che)R and CheB molecules are approximately 500- and 160-fold higher in S. meliloti than in E. coli, respectively. Since not all chemoreceptors in chemotactic bacteria have and utilize the pentapeptide moiety, we investigated the S. meliloti system and observed a strong interaction between CheR, activated CheB and the isolated pentapeptides via in-vitro binding studies. On the contrary, unmodified CheB showed weak binding to the pentapeptide. Through in-vivo studies, we highlighted the physiological necessity of the pentapeptide for chemotaxis. S. meliloti strains with substitutions of the conserved tryptophan residue to alanine in one or all four pentapeptide-bearing Methyl-accepting Chemotaxis Proteins (MCPs) resulted in diminished or loss of chemotaxis to glycine betaine, lysine, and acetate, ligands sensed by pentapeptide-bearing McpX and pentapeptide-lacking McpU and McpV, respectively. The flexible linker connecting the pentapeptide to the MCPs together with the pentapeptide itself were shown to be functional on pentapeptide-lacking chemoreceptors and provided adaptational assistance to other chemoreceptors that lacked a functional pentapeptide. Based on these results, we concluded that S. meliloti employs a pentapeptide-dependent adaptation system with MCPs possessing a consensus pentapeptide motif (N/D)WE(E/N)F). Finally, we postulated that the higher abundance of CheR and CheB in S. meliloti compared to E. coli compensates for the lower number of pentapeptide-bearing chemoreceptors in the chemosensory array. In chapter 3, we explored the putative phosphatase function of a novel protein, CheT, on phosphorylated S. meliloti response regulators. The kinase CheA phosphorylates both the sink response regulator, CheY1, and the flagellar motor interacting response regulator, CheY2. CheY1 competes with CheY2 for these phosphate groups, but we have discovered another layer of complexity to the story. Sequence comparison of S. meliloti CheT and the E. coli phosphatase CheZ shows little sequence homology. However, both proteins share a DXXXQ phosphatase motif. Phosphorylation assays performed using radiolabeled [γ-32P]-ATP revealed that CheT acts as a phosphatase of CheY1~P and accelerates dephosphorylation of CheY1~P by at least two-fold. Interestingly, we also discovered that CheT interacts with CheR, but this interaction did not affect the enzymatic activity of either protein under the examined conditions. Unexpectedly, a cheT deletion strain and strains carrying mutations in the phosphatase motif exhibit an increased swimming speed, a phenotype that does not conform with the model that the absence of CheT or its activity results in increased CheY2~P levels and reduced swimming speed. We concluded that a revised S. meliloti signal termination pathway should include CheT enhancing dephosphorylation of CheY1~P and sensory adaptation involving the yet unknown function of CheT on CheR. While the adaptation system in S. meliloti is unexplored, this work provides first insights into fascinating deviations and similarities to the known paradigm. We have also delivered evidence that the S. meliloti signal termination system requires a dedicated phosphatase. The knowledge gained here takes us a step closer to enhance the S. meliloti chemotaxis pathway towards improved symbiosis with alfalfa and to reduce our dependence on environmentally deleterious synthetic fertilizers. / Doctor of Philosophy / Like all living things, bacteria inhabit a constantly changing environment, hence the need to take up and process this information. Bacterial cells have evolved sophisticated biological tools to tackle this challenge of detecting, responding and adapting to environmental signals like nutrients, toxins, temperature changes, light, metabolites, etc. Motile bacteria such as Escherichia coli, a gut resident microbe, and Sinorhizobium meliloti, a soil dwelling bacterium, direct their swimming behavior in response to chemical gradients within the milieu through a process termed chemotaxis. Generally, this vital process enables a bacterium to escape harmful chemicals and gravitate towards beneficial ones. However, S. meliloti specifically employs chemotaxis to locate the roots of its plant host (alfalfa) and to establish a symbiotic relationship through which the bacteria provide essential nitrogen for plant growth in exchange for nourishment. The biological tools employed by S. meliloti for chemotaxis include environmental sensing receptors called Methyl-accepting Chemotaxis Proteins (MCPs) and proteins inside the bacterial cell that transfer information from the sensors to long, helical rotating propeller structures, called flagella. Importantly, the efficiency of this process hinges on a timely termination of information flow and the ability to adapt to prevailing stimuli while maintaining sensitivity to increasing concentration gradients. This work investigates the function of the C-terminal five amino acid motif of MCPs known to be critical for adaptation in E. coli and the phosphatase activity of a novel protein, CheT, in signal termination of S. meliloti chemotaxis system.

Traditional multiple hypothesis testing procedures, such as that of Benjamini and Hochberg, fix an error rate and determine the corresponding rejection region. In 2002 Storey proposed a fixed rejection region procedure and showed numerically that it can gain more power than the fixed error rate procedure of Benjamini and Hochberg while controlling the same false discovery rate (FDR). In this thesis it is proved that when the number of alternatives is small compared to the total number of hypotheses, Storeys method can be less powerful than that of Benjamini and Hochberg. Moreover, the two procedures are compared by setting them to produce the same FDR. The difference in power between Storeys procedure and that of Benjamini and Hochberg is near zero when the distance between the null and alternative distributions is large, but Benjamini and Hochbergs procedure becomes more powerful as the distance decreases. It is shown that modifying the Benjamini and Hochberg procedure to incorporate an estimate of the proportion of true null hypotheses as proposed by Black gives a procedure with superior power.<p> Multiple hypothesis testing can also be applied to regression diagnostics. In this thesis, a Bayesian method is proposed to test multiple hypotheses, of which the i-th null and alternative hypotheses are that the i-th observation is not an outlier versus it is, for i=1,...,m. In the proposed Bayesian model, it is assumed that outliers have a mean shift, where the proportion of outliers and the mean shift respectively follow a Beta prior distribution and a normal prior distribution. It is proved in the thesis that for the proposed model, when there exists more than one outlier, the marginal distributions of the deletion residual of the i-th observation under both null and alternative hypotheses are doubly noncentral t distributions. The outlyingness of the i-th observation is measured by the marginal posterior probability that the i-th observation is an outlier given its deletion residual. An importance sampling method is proposed to calculate this probability. This method requires the computation of the density of the doubly noncentral F distribution and this is approximated using Patnaiks approximation. An algorithm is proposed in this thesis to examine the accuracy of Patnaiks approximation. The comparison of this algorithms output with Patnaiks approximation shows that the latter can save massive computation time without losing much accuracy.<p> The proposed Bayesian multiple outlier identification procedure is applied to some simulated data sets. Various simulation and prior parameters are used to study the sensitivity of the posteriors to the priors. The area under the ROC curves (AUC) is calculated for each combination of parameters. A factorial design analysis on AUC is carried out by choosing various simulation and prior parameters as factors. The resulting AUC values are high for various selected parameters, indicating that the proposed method can identify the majority of outliers within tolerable errors. The results of the factorial design show that the priors do not have much effect on the marginal posterior probability as long as the sample size is not too small.<p> In this thesis, the proposed Bayesian procedure is also applied to a real data set obtained by Kanduc et al. in 2008. The proteomes of thirty viruses examined by Kanduc et al. are found to share a high number of pentapeptide overlaps to the human proteome. In a linear regression analysis of the level of viral overlaps to the human proteome and the length of viral proteome, it is reported by Kanduc et al. that among the thirty viruses, human T-lymphotropic virus 1, Rubella virus, and hepatitis C virus, present relatively higher levels of overlaps with the human proteome than the predicted level of overlaps. The results obtained using the proposed procedure indicate that the four viruses with extremely large sizes (Human herpesvirus 4, Human herpesvirus 6, Variola virus, and Human herpesvirus 5) are more likely to be the outliers than the three reported viruses. The results with thefour extreme viruses deleted confirm the claim of Kanduc et al.

Traditional multiple hypothesis testing procedures, such as that of Benjamini and Hochberg, fix an error rate and determine the corresponding rejection region. In 2002 Storey proposed a fixed rejection region procedure and showed numerically that it can gain more power than the fixed error rate procedure of Benjamini and Hochberg while controlling the same false discovery rate (FDR). In this thesis it is proved that when the number of alternatives is small compared to the total number of hypotheses, Storeys method can be less powerful than that of Benjamini and Hochberg. Moreover, the two procedures are compared by setting them to produce the same FDR. The difference in power between Storeys procedure and that of Benjamini and Hochberg is near zero when the distance between the null and alternative distributions is large, but Benjamini and Hochbergs procedure becomes more powerful as the distance decreases. It is shown that modifying the Benjamini and Hochberg procedure to incorporate an estimate of the proportion of true null hypotheses as proposed by Black gives a procedure with superior power.<p> Multiple hypothesis testing can also be applied to regression diagnostics. In this thesis, a Bayesian method is proposed to test multiple hypotheses, of which the i-th null and alternative hypotheses are that the i-th observation is not an outlier versus it is, for i=1,...,m. In the proposed Bayesian model, it is assumed that outliers have a mean shift, where the proportion of outliers and the mean shift respectively follow a Beta prior distribution and a normal prior distribution. It is proved in the thesis that for the proposed model, when there exists more than one outlier, the marginal distributions of the deletion residual of the i-th observation under both null and alternative hypotheses are doubly noncentral t distributions. The outlyingness of the i-th observation is measured by the marginal posterior probability that the i-th observation is an outlier given its deletion residual. An importance sampling method is proposed to calculate this probability. This method requires the computation of the density of the doubly noncentral F distribution and this is approximated using Patnaiks approximation. An algorithm is proposed in this thesis to examine the accuracy of Patnaiks approximation. The comparison of this algorithms output with Patnaiks approximation shows that the latter can save massive computation time without losing much accuracy.<p> The proposed Bayesian multiple outlier identification procedure is applied to some simulated data sets. Various simulation and prior parameters are used to study the sensitivity of the posteriors to the priors. The area under the ROC curves (AUC) is calculated for each combination of parameters. A factorial design analysis on AUC is carried out by choosing various simulation and prior parameters as factors. The resulting AUC values are high for various selected parameters, indicating that the proposed method can identify the majority of outliers within tolerable errors. The results of the factorial design show that the priors do not have much effect on the marginal posterior probability as long as the sample size is not too small.<p> In this thesis, the proposed Bayesian procedure is also applied to a real data set obtained by Kanduc et al. in 2008. The proteomes of thirty viruses examined by Kanduc et al. are found to share a high number of pentapeptide overlaps to the human proteome. In a linear regression analysis of the level of viral overlaps to the human proteome and the length of viral proteome, it is reported by Kanduc et al. that among the thirty viruses, human T-lymphotropic virus 1, Rubella virus, and hepatitis C virus, present relatively higher levels of overlaps with the human proteome than the predicted level of overlaps. The results obtained using the proposed procedure indicate that the four viruses with extremely large sizes (Human herpesvirus 4, Human herpesvirus 6, Variola virus, and Human herpesvirus 5) are more likely to be the outliers than the three reported viruses. The results with thefour extreme viruses deleted confirm the claim of Kanduc et al.