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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Structural Studies of the Klebsiella Pneumoniae Pantothenate Kinase in Complex with Pantothenamide Substrate Analogues

Li, Buren 20 November 2012 (has links)
N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.
2

Structural Studies of the Klebsiella Pneumoniae Pantothenate Kinase in Complex with Pantothenamide Substrate Analogues

Li, Buren 20 November 2012 (has links)
N-substituted pantothenamides are analogues of pantothenate, the precursor of the essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive target for drug discovery due to low sequence homology between bacterial and human PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is located within a highly aromatic pocket, suggesting that an aromatic substituent may enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved. The structure reveals that the pyridine ring adopts alternative conformations in the aromatic pocket, providing the structural basis for further improvement of pantothenamide-binding to KpPanK.
3

Developing Novel Electrospray Ionization Mass Spectrometry (esi ms) Techniques to Study Higher Order Structure and Interaction of Biopolymers

Frimpong, Agya K. 01 September 2009 (has links)
Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

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