Fundamental Studies of Enzymes: From Single Molecule Kinetics to Conformational Studies.

摘要

This thesis covers the usage of the fiber arrays developed in our lab for sensitive detection of individual molecules. We were able to apply this platform to study activities and conformational changes of individual enzymes. For this purpose we developed a heating stage which we were able to apply for heating studies described in following chapters.;In chapter 1 we presented the current detection approach of studying single enzymes. Although great amount of new techniques were developed recently due to advance in technology, we focused only on fluorescent based studies. Moreover in the chapter 1 we cover only the general approach and any detailed descriptions of currently used fluorescent techniques would be impossible to cover within this thesis.;Chapter 2 describe in detail the development and the basic principles of the platform used in our single molecule research. In addition to previously described fiber bundles and sealing techniques we implemented the heating stage. The heating platform allows us to study single enzymes with respect to temperature variations. Precise control of the temperature during the experiment allows us to measure the activities of enzymes at different temperatures as well to study conformation changes and denaturation.;In Chapter 3 we tried to unearth origins of the stable heterogeneity of the activity of individual enzymes. We performed the heating pulse experiment and we have found that the activities of individual enzymes change after heating pulse, where the average activity stays constant. From this experiment we concluded that the conformation needs to play the leading role in the distribution in activity.;In Chapter 4 we used the temperature ramp to calculate the activation energies of individual enzymes. We were able to monitor the activities of individual &;In Chapter 5 we performed single molecule experiments on the diferent &;Finally Chapter 6 describes the thermal denaturation of monomeric enzyme and refolding studies performed on the single molecule level. Gaining a more fundamental understanding of how individual enzymes fold and work within a population should provide insight into how they affect downstream biochemical processes as well how the folding and misfolding process occur.