Structure and dynamics of the mesodomain environment of coenzyme B12-dependent ethanolamine ammonia-lyase in frozen aqueous solutions and kinetics of the radical rearrangement reaction at 173-187 K Open Access

Chen, Hanlin (2014)

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The aqueous solution environment can has been proposed to control influence protein function by coupling with protein functions through bulk solvent motions and hydration-shell fluctuations. To provide a deeper understanding of the relative importance of solvent mobility, intrinsic protein motions and coupling between solvent and protein motions, this dissertation embarks on characterizing the properties of the mesodomains in aqueous sucrose solutions over the range from 0 - 75% (w/v) of added sucrose with , by using the paramagnetic probe, TEMPOL, and by using multiple EPR electron paramagnetic resonance (EPR) techniques. The results provide a detailed view characterize the glass transition, TEMPOL concentration, and volume of the of the macro-scale solute-solvent structure in frozen sucrose solutionsmesodomain., and insights about their interactions under aqueous conditions These studies are extended to investigate the microscopic structure of frozen aqueous sucrose solutions [0 - 75% (w/v)], which contain the coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium, with and without the substrate, ethanolamine. It is concluded that in frozen aqueous solutions, the protein creates a mesodomain in frozen aqueous solutions which displays different properties in structure and dynamics from the polycrystalline bulk solvent. To In a separate study, the protein "glass" transition, the kinetics decay of the Co(II)-substrate radical pair decay reaction were measured at four ultra low temperatures of 173-187 K, which are below the mesodomain glass transition. (173, 177, 183 and 187 K) was investigated. The power-law decay kinetics indicate the emergence of multiple exponential phases, suggesting that protein, and possibly coupled non-bulk, hydration solvent, motions become rate determining for the radical rearrangement in the examined temperature range. Overall, the mesodomain and low temperature kinetic studies contribute to the understanding of the roles of protein and coupled solvent dynamics in EAL function.

Table of Contents

Table of Contents

Chapter 1: Introduction 1

1.1 Coenzyme B12 -dependent enzyme, ethanolamine ammonia-lyase (EAL) 2

1.2 Structure of B12 4

1.3 Minimal mechanism of catalysis for EAL 5

1.4 EPR studies on the kinetics of EAL catalyzed Co(II)-substrate radicals 7

1.5 The "glass" transition of proteins and the role of solvent 7

1.6 Continuous-wave electron paramagnetic resonance (EPR) spectroscopy 9

1.7 Electron spin echo envelope modulation (ESEEM) spectroscopy 15

1.8 Outline of Dissertation 18

Chapter 2: Heterogeneous Ordered-Disordered Structure of the Mesodomain in Frozen Sucrose-Water Solutions Revealed by Multiple Electron Paramagnetic Resonance Spectroscopies 19

2.1 Background and introduction 20

2.1.1 Mesodomain and temperature-composition (T-c) diagram 20

2.1.2 Sample preparations and experimental setup 23

2.2 Experimental results 27

2.2.1 Mesodomain Mobility transition of the TEMPOL paramagnetic probe observed from EPR lineshape analysis 27

2.2.2 Relative sucrose concentration in the mesodomain from electron spin echo envelope modulation (ESEEM) spectroscopy 31

2.2.3 Relative TEMPOL mesophase concentration from spin-lattice relaxation time in sucrose solutions 34

2.2.4 Calibration of TEMPOL concentration with spin-lattice relaxation time in glassy 60% (w/v) sucrose solution 37

2.2.5 Relative TEMPOL mesodomain concentration from phase memory relaxation time in aqueous sucrose solutions 38

2.3 Probing the various perspectives of the mesodomain 40

2.3.1 Identification of the mesodomain and properties in pure water and 1% (w/v) sucrose solution 40

2.3.2 Relative volume of the mesodomain in unsaturated sucrose-water solutions 41

2.3.3 Spin probe concentration in the mesodomain 43

2.3.4 Heterogeneous structure of the mesodomains formed from unsaturated sucrose-water solutions 44

2.4 Conclusion 47

Chapter 3: The Frozen Solution Environment of ethanolamine ammonia-lyase (EAL) and its substrate ethanolamine detected by electron paramagnetic resonance spectroscopies 48

3.1 Background and Introduction 49

3.2 Sample preparation and experimental setups 51

3.3 Local environment mobility from CW-EPR spectra 56

3.4 Local sucrose concentration revealed by the envelope modulation depth (EMD) analysis from Electron Spin-Echo Envelope Modulation (ESEEM) spectroscopy 59

3.5 Longitudinal relaxation times (T1) obtained from repetition dependence of ESE measurements to reveal the relative TEMPOL concentration 62

3.6 Phase memory times (TM) obtained from 2-pulse ESEEM spectra analysis to reveal the relative TEMPOL concentration 67

3.7 Discussion 70

3.7.1 Identification of the mesodomain and properties in sucrose solutions with EAL or EAL/ethanolamine 70

3.7.2 Relative volume of the mesodomain in sucrose solutions with EAL or EAL/ethanolamine 71

3.7.3 Heterogeneous structures of the mesodomains formed in sucrose solutions with EAL or EAL/ethanolamine 72

3.8 Conclusion 73

Chapter 4: Probing Decay Kinetics of Co(II)-[1H]-substrate radical pair at temperatures below 190 K 75

4.1 Introduction 76

4.2 Sample preparation and time-resolved EPR measurements 79

4.3 Results and discussion 81

4.4 Conclusion 91

Chapter 5: Connections: EPR spectroscopic and relaxation studies of the solution and protein mesodomain environments in frozen aqueous solutions, and kinetics of the radical arrangement reaction in EAL 92

Bibliog r aphy 97

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