Enzyme Dynamics Elucidated via Temperature Jump Fluorescence Spectroscopy Pubblico

Abstract

Enzymes are nature’s catalyst. They drastically increase rates of reactions that are necessary for all living organisms. In order to take full advantage of the potential of enzymes to solve medical, environmental, and industrial challenges, we must fully understand how they function. Enzymes are dynamic molecules that move and change conformations, yet many models used to manipulate and predict enzymes treat them as static structures. We must identify motions across all timescales and determine their function so that enzyme dynamics can be incorporated into our models. The microsecond time regime is traditionally difficult to access and thus, has not been well studied. Laser-induced temperature jump fluorescence spectroscopy can measure events occurring on the tens of nanoseconds out to several milliseconds, encompassing the entirety of the microsecond time regime. Herein, we focus on enzyme dynamics on the microsecond timescale such as loop motions and collective motions that impact the search for reactive conformations. We use temperature jump fluorescence spectroscopy to elucidate the dynamics of two enzyme systems: Escherichia colidihydrofolate reductase and Bacillus stearothermophilus alcohol dehydrogenase. With dihydrofolate reductase we examined both global and local motions in addition to measuring the transition rates of an important active site loop. With alcohol dehydrogenase we discovered collective motions that are responsible for anomalous temperature dependent behavior. Together, these studies provide a better understanding of enzyme dynamics as well as a framework for future investigations.

Table of Contents

Chapter 1: Introduction ............................................................................................................ 1

1.1 Enzyme Dynamics and Function ............................................................................................... 1

1.2 Dihydrofolate Reductase ............................................................................................................. 4

1.3 Alcohol Dehydrogenase .............................................................................................................. 7

1.4 Temperature Jump Spectroscopy ............................................................................................. 10

1.5 References .................................................................................................................................... 14

Chapter 2: Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase ...... 18

2.1 Abstract ........................................................................................................................................ 18

2.2 Introduction ................................................................................................................................ 19

2.3 Experimental Procedures .......................................................................................................... 22

2.4 Results and Discussion .............................................................................................................. 26

2.5 Supplemental Information ........................................................................................................ 40

2.6 References .................................................................................................................................... 46

Chapter 3: Site-specific Tryptophan Labels Reveal Local Millisecond Motions of Dihydrofolate Reductase ....................................................................................................... 52

3.1 Abstract ........................................................................................................................................ 52

3.2 Introduction ................................................................................................................................ 53

3.3 Experimental Methods .............................................................................................................. 54

3.4 Results and Discussion .............................................................................................................. 56

3.5 Conclusion ................................................................................................................................... 64

3.6 Supplemental Information ........................................................................................................ 65

3.7 References .................................................................................................................................... 68

Chapter 4: Viscosity Dependent Met20 Loop Motions of Dihydrofolate Reductase ............ 70

4.1 Abstract ........................................................................................................................................ 70

4.2 Introduction ................................................................................................................................ 71

4.3 Experimental Methods .............................................................................................................. 72

4.4 Results and Discussion .............................................................................................................. 74

4.5 Conclusion ................................................................................................................................... 87

4.6 Supplemental Information ........................................................................................................ 88

4.7 References .................................................................................................................................... 89

Chapter 5: Activity-Related Microsecond Dynamics Revealed by Temperature-Jump Förster Resonance Energy Transfer Measurements on Thermophilic Alcohol Dehydrogenase ..... 92

5.1 Abstract ........................................................................................................................................ 92

5.2 Introduction ................................................................................................................................ 93

5.3 Results and Discussion .............................................................................................................. 94

5.4 Supplemental Information ...................................................................................................... 100

5.5 References .................................................................................................................................. 111

Chapter 6: Conclusion .......................................................................................................... 114

6.1 Alcohol Dehydrogenase .......................................................................................................... 114

6.2 Dihydrofolate Reductase ......................................................................................................... 115

6.3 Perspective ................................................................................................................................. 118

6.4 References .................................................................................................................................. 119

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Chemistry, Physical Chemistry, Biochemistry
Parola chiave	Dihydrofolate Reductase Dynamics Enzyme Alcohol Dehydrogenase Protein Motions
Committee Chair / Thesis Advisor	Dyer, Brian, Emory University
Committee Members	Conticello, Vincent, Emory University Lutz, Stefan, Emory University

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