The research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study.... Show moreThe research aims to explore the evolutionary adaptability of enzymes and the impact of temperature on protein evolution pathways, using M. tuberculosis β-lactamase BlaC as the object of study. Enzymes inherently embody a delicate balance between activity and stability, and the acquisition of new enzymatic functions is often accompanied by trade-offs, such as decreased stability or reduction of the original activity. Probing evolutionary adaptability of BlaC with laboratory evolution in combination with structural characterization can provide information about the mechanisms of rapid adaptations observed for β-lactamases in the clinic. The role of temperature as a conventional selection pressure in such evolutionary adaptation is unclear. The cooperative nature of enzyme unfolding over a narrow temperature trajectory raises the question whether evolution at temperatures well below the melting point is influenced by temperature. The approach used in this work to answer these questions is by simulating evolution under different selection pressures and characterize the variant enzymes in terms of activity, structure, dynamics and melting temperature. The research makes clear how enzyme kinetics and dynamics vary with different selection pressures and maps the evolutionary path that enzymes may take. The underlying structural mechanisms are established to provide a rationale for the observed effects. Show less
Ever since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and... Show moreEver since the structural data of biological macromolecules became available, there has been consistent struggle to relate this new information to the existing spectroscopy, activity and theoretical descriptions of these proteins and to understand the evolution and/or to predict the role of yet uncharacterized gene products in this light. The research presented in this thesis primarily deals with understanding the structure__function relationship of a newly discovered blue copper protein. The protein is derived from Streptomyces coelicolor and is called small laccase (SLAC). It utilizes four copper ions to catalyze the oxidation of substrate molecules concomitant with the reduction of oxygen to water. The catalytic cycle of this enzyme is studied using a variety of spectroscopic and kinetic methods in an attempt to improve our understanding of the internal operations which are critical to its functioning. The new results obtained are presented in this thesis. Show less
This thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact... Show moreThis thesis centers around a novel fluorescence based method that allows to monitor the activity of redox enzymes and of electron (ET) or oxygen transfer proteins. It takes advantage of the fact that the absorption spectrum of the protein__s active site varies upon changing its redox state. This change can be translated into a change in the fluorescence intensity of a label that is covalently linked to the protein on the basis of F_rster Resonance Energy Transfer (FRET). With our method we could show that different redox proteins and enzymes can be studied down to the single molecule level. This exciting finding opens the door to the study of various redox enzymes and to monitor specific substances such as for example nitrite. Depending on the function of the enzyme under investigation a wide range of substrates can be monitored. Another example is the development of an oxygen sensor by employing proteins that are capable of binding oxygen. The findings presented in this thesis might be significant for applications in oxygen sensing and, more generally, in the fast growing field of biosensing Show less
