X-ray crystallography is used as a powerful tool to understand how a particular biomacromolecule accomplishes its various functions. In the present study, detailed atomic structure of bacterial di-heme cytochrome c4 isolated for the first time from an anaerobic organism and structure of two crystal forms of sweet protein thaumatin from the African shrub, are presented, as well as an example of how this structural knowledge of cytochrome c4 in combination with biochemical data have been used to classified the protein function. In parallel to modern high-throughput approaches in the crystallization of protein, basic research on their physico-chemical properties and molecular interactions has increasingly gained on importance in recent years. Our aim was to improve the success of any crystallization attempts significantly as well as to find alternative methods of predicting it. In this thesis two novel approaches in macromolecular crystallization are discussed. The first; newly discovered cross-crystallization method alias cross-influence procedure (CIP) with its subsequent application presented on two selected proteins. And the novelty in our second approach is the use of precise temperature-control to drive the crystallization process by keeping system in metastable zone. The understanding that the solubility and phase behavior of proteins is an essential part of the crystallization process, allows us to perform the temperature-controlled experiment (TCE), where the manipulation of the thermodynamics as well as the kinetics of the crystallization process were controlled. In order to improve crystal quality and/or morphology, the both CIP and TCE were found as useful tools in protein crystallization presented by means of crystallization of three different proteins (bacterial cyrochrome c4, glycoside hydrolase family 11 enzyme xylanase and sweet protein thaumatin). Finally, the preliminary crystallization information about three other enzymes (two haloalkane dehalogenase and one membrane protein hydrogenase) are given at the end, as a starting experiment for further X-ray diffraction analysis.
| Identifer | oai:union.ndltd.org:nusl.cz/oai:invenio.nusl.cz:80489 |
| Date | January 2007 |
| Creators | TOMČOVÁ, Ivana |
| Source Sets | Czech ETDs |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/restrictedAccess |
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