Fundamental Studies of Enzymes: From Single Molecule Kinetics to Conformation Studies.
Abstract: 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 curr... read moreent 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
β-galactosidase enzymes with respect to increased temperature. In addition to
activation energy we were able to obtain the entropy and enthalpy values by using the
Eyring-Polanyi equation. We showed that the activation energy greatly distributes along the
molecules and enzymes with different thermal stabilities might be present within the
population. In Chapter 5 we performed single molecule experiments on the diferent
β-glucuronidase mutants. By using the heating platform we demonstrate that at the
single molecule level a fraction of the individual enzymes behave as thermal switches,
where they lack activity at room temperature but become active when treated with a heating
pulse. Those "thermal switches" could be beneficial for developing specific drug carriers,
where a thermal pulse would trigger downstream enzymatic reactions. 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
Thesis (Ph.D.)--Tufts University, 2015.
Submitted to the Dept. of Chemistry.
Advisor: David Walt.
Committee: Krishna Kumar, Elena Rybak-Akimova, and Dagmar Ringe.
Keyword: Chemistry.read less