Toward Connecting Solvent and Surface Dynamics to Protein Function: An EPR Approach Open Access
Nforneh, Benjamen (Summer 2018)
Abstract
The 12B (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Electron paramagnetic resonance (EPR) spectroscopy of the spin probe, TEMPOL, is used to resolve two distinct concentric solvent phases around EAL: protein-associated domain (PAD) and mesodomain. By using a continuum model, we estimate the solvent shell thicknesses around EAL (mesodomain ~ 10-40 Å and PAD ~ 6 Å) and propose a model for the system. Systematic tuning of solvent dynamics and glass transitions by using dimethylsulfoxide (DMSO) variation in the low-T mesodomain system reveals features of the mesodomain/PAD and PAD/protein surface coupling that inform the understanding of solvent and coupled protein dynamics to chemical catalysis in EAL. Toward understanding the function of the EutC N-terminal, signal sequence associated domain of wild type EAL, and the interplay between protein stability and the EAL targeting and trafficking to the Eut biomicrocompartment (BMC), spin label 4-maleimido-TEMPO (4MT) attached at the C37 of EutC and EPR spectroscopy is used. A two-state model is propose based on a single 4MT labelling site, in which fast and slow motional populations represent EAL bound and free conformations of the EutC N-terminal domain. We propose that the two states present a balance between EutC function and EAL protein stability and efficient targeting to the BMC. The observed fluidizing effect of the added % v/v DMSO on the dynamics in the mesodomain, and the coupled increase in PAD dynamics, is manifested in the lowering of the fluid-solid transitions in each phase. This provides a method to precisely control the solvent and surface dynamics around EAL as a tunable parameter in quantifying and investigating the mechanism of coupling between solvent and surface dynamics, and chemical reaction steps in EAL.
Table of Contents
Distribution Agreement i
Acknowledgments vi
List of Figures vii
List of Tables xv
Abbreviations xix
1 Introduction and Background, Technique, and Overview 1
1.1 INTRODUCTION AND BACKGROUND . . . . . . . . . . . . . . . 2
1.1.1 Structure of B12 coenzyme adenosylcobalamin
(AdoCbl) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
1.1.2 The ethanolamine ammonia-lyase enzyme: Reaction and
mechanism of action . . . . . . . . . . . . . . . . . . . . . . . 7
1.1.3 The ethanolamine utilization bacterial microcompactment
and the role of ethanolamine ammonia-lyase . . . . . . . . . 11
1.2 EXPERIMENTAL TECHNIQUE . . . . . . . . . . . . . . . . . . . . . 13
1.2.1 Electron paramagnetic resonance spectroscopy . . . . . . . . 13
1.2.2 Generation of an electron paramagnetic resonance spectrum 22
1.3 OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2 Mesodomain and Protein-Associated Solvent Phases with Temperature-
Tunable (200-265 K)
Dynamics Surround Ethanolamine Ammonia-Lyase in Globally Polycrystalline
Aqueous Solution Containing Dimethylsulfoxide 26
2.1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . . . . . . 30
2.2.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.2 Continuous-wave EPR spectroscopy . . . . . . . . . . . . . . 31
2.2.3 EPR Simulations . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.1 Temperature dependence of the TEMPOL EPR line shape
in frozen aqueous solution with EAL: 0% DMSO . . . . . . . 34
2.3.2 Temperature dependence of the TEMPOL EPR line shape
in frozen aqueous solution with EAL: 1% v/v DMSO . . . . 36
2.3.3 Temperature dependence of the TEMPOL rotational correlation
times and normalized component weights in frozen
aqueous solution with EAL: 0% DMSO . . . . . . . . . . . . 36
2.3.4 Temperature dependence of the TEMPOL rotational correlation
times and normalized component weights in frozen
aqueous solution with EAL: 1% v/v DMSO . . . . . . . . . . 39
2.3.5 Temperature dependence of the TEMPOL rotational correlation
times and normalized component weights in the absence
of EAL: 0 and 1% v/v DMSO . . . . . . . . . . . . . . 41
2.3.6 EAL protein concentration dependence of the EPR line shape
in frozen 1% v/v DMSO solution . . . . . . . . . . . . . . . . 44
2.4 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.4.1 Origin of the mobility components in the EAL, 0% DMSO
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.4.2 Origin of the mobility components in the EAL, 1% v/v DMSO
condition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.4.3 Mobility transition in the EAL, 1% v/v DMSO system . . . 49
2.4.4 Behavior at T-values above the mobility transition in the
EAL, 1% v/v DMSO system . . . . . . . . . . . . . . . . . . . 51
2.4.5 Relative volumes of the PAD and mesodomain . . . . . . . . 51
2.4.6 Origin of the temperature-dependence of TEMPOL tumbling
mobility . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.5 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3 Ice Boundary and Protein Suface Confinement Effects Govern Proteinassociated
and Mesodomain Solvent Dynamics Around the B12-Dependent
Ethanolamine Ammonia-Lyase Protein in Frozen Aqueous-Dimethylsulfoxide
Solutions 59
3.1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
3.2 EXPERIMENTAL METHODS . . . . . . . . . . . . . . . . . . . . . . 63
3.2.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2.2 Continuous wave EPR spectroscopy and EPR Simulations . 64
3.3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3.1 Temperature dependence of the TEMPOL EPR line shape
in frozen aqueous solution with EAL: 0.5, 2.0 and 4.0% v/v
DMSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
3.3.2 Temperature dependence of the TEMPOL rotational correlation
times and normalized component weights in frozen
aqueous solution with EAL: 0.5, 2.0 and 4.0% v/v DMSO . . 67
3.3.3 Temperature dependence of the TEMPOL spectral line
shape in the absence of EAL: 0.5, 2.0 and 4.0% v/v DMSO . 69
3.3.4 Temperature dependence of the TEMPOL rotational correlation
times and normalized component weights in solution,
in the absence of EAL: 0.5, 2.0 and 4.0% v/v DMSO . . 71
3.4 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
3.4.1 Added DMSO resides predominantly in the mesodomain . 72
3.4.2 Relative dimensions of mesodomain, PAD and EAL . . . . . 75
3.4.3 Resolution of an order-disorder transition in the proteinassociated
domain . . . . . . . . . . . . . . . . . . . . . . . . 77
3.4.4 Composition and fluidity of the mesodomain . . . . . . . . . 79
3.4.5 Temperature-dependence of spin probe mobility in mesodomain
and PAD in Region III . . . . . . . . . . . . . . . . . . . . . . 80
3.4.6 Combination of protein and ice boundary confinement effects
lead to DMSO-concentration–dependence of solvent
mobility and the order-disorder transition in the PAD . . . . 82
3.4.7 Dependence of mesodomain solvent dynamics on PAD solidification
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.5 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4 Electron Spin-Labeling of the EutC Subunit in B12-Dependent Ethanolamine
Ammonia-Lyase Reveals Dynamics and a Two-State Conformational Equilibrium
in the N-terminal, Signal-Sequence-Associated Domain 91
4.1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4.2 MATERIAL AND METHODS . . . . . . . . . . . . . . . . . . . . . . 95
4.2.1 Protein preparation . . . . . . . . . . . . . . . . . . . . . . . . 95
4.2.2 EPR sample preparation . . . . . . . . . . . . . . . . . . . . . 95
4.2.3 Site-directed mutagenesis . . . . . . . . . . . . . . . . . . . . 96
4.2.4 Continuous wave EPR spectroscopy . . . . . . . . . . . . . . 99
4.2.5 Continuous wave EPR simulations . . . . . . . . . . . . . . . 100
4.2.6 Criteria for detection of spin label motion . . . . . . . . . . . 101
4.3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
4.3.1 Accessibility of cysteines in wt EAL . . . . . . . . . . . . . . 102
4.3.2 Identification of the 4MT labeling site in wt EAL . . . . . . . 103
4.3.3 Temperature dependence of the 4MT-labeled EAL EPR line
shape in frozen aqueous solution, in the absence of DMSO . 106
4.3.4 Temperature dependence of the 4MT-labeled EAL EPR line
shape in frozen aqueous solution with 1% v/v DMSO . . . . 108
4.3.5 Temperature dependence of the 4MT-labeled EAL rotational
correlation time and normalized component weights in frozen
aqueous solution, in the absence of DMSO . . . . . . . . . . 108
4.3.6 Temperature dependence of the 4MT-labeled EAL rotational
correlation time and normalized component weights in frozen
aqueous solution with 1% v/v DMSO . . . . . . . . . . . . . 110
4.4 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
4.4.1 Nature of the primary betaC37 spin-labeling site in wt EAL . . 112
4.4.2 Two-state model for 4MT mobility in EAL in frozen solution
in the absence and presence of DMSO (0 and 1% v/v
DMSO) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
4.4.3 Solvent- and temperature-dependence of the populations
in the two-state system . . . . . . . . . . . . . . . . . . . . . . 116
4.4.4 Equilibrium between the states, Ss and Sf . . . . . . . . . . . 116
4.4.5 Model for the origin and temperature-dependence of the
two-state, Ss, Sf system for EAL in 0 and 1% DMSO solution 119
5 The Two-State Conformational Equilibrium in the N-terminus of the
EutC Subunit of EAL Revealed by using Electron Spin-Labeling is Maintained
in Frozen added 0.5, 2 and 4% v/v Dimethylsulfoxide-Water Solution
123
5.1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
5.2 MATERIAL AND METHODS . . . . . . . . . . . . . . . . . . . . . . 126
5.2.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 126
5.2.2 Continuous wave EPR spectroscopy . . . . . . . . . . . . . . 127
5.2.3 Continuous wave EPR simulations and criteria for detection
of spin label motion . . . . . . . . . . . . . . . . . . . . . 127
5.3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
5.3.1 Temperature dependence of the 4MT-labeled EAL EPR line
shape in frozen aqueous solution: 0.5, 2 and 4% v/v DMSO 128
5.3.2 Temperature dependence of the 4MT-labeled EAL rotational
correlation times and normalized component weights in frozen
aqueous solution with 0.5, 2 and 4% v/v DMSO . . . . . . . 130
5.4 DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
5.4.1 Origin of the 4MT mobility components in the spin-labeled
EAL aqueous solution: 0.5, 2 and 4% v/v DMSO systems . . 132
5.4.2 Confirmation of an existing equilibrium between the states,
Ss and Sf in 0.5, 2 and 4% v/v DMSO systems . . . . . . . . 133
5.4.3 Temperature dependence of the rigid to mobile dynamic
transition and the role of the added DMSO . . . . . . . . . . 135
6 The Effects of added Substrate, Aminoethanol, on the Solvent Dynamics
Around the B12-Dependent Ethanolamine Ammonia-Lyase Protein in
Frozen Aqueous-Dimethylsulfoxide Solution 138
6.1 INTRODUCTION AND BACKGROUND . . . . . . . . . . . . . . . 139
6.2 MATERIAL AND METHODS . . . . . . . . . . . . . . . . . . . . . . 142
6.2.1 Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . 142
6.2.2 Continuous wave EPR spectroscopy and EPR Simulations . 142
6.3 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 143
6.3.1 Temperature dependence of the TEMPOL EAL EPR line
shape in frozen aqueous solution with AmEtOH: 0, and 2%
v/v DMSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
6.3.2 Temperature dependence of the TEMPOL EAL rotational
correlation times and normalized component weights in frozen
aqueous solution with AmEtOH: No DMSO . . . . . . . . . 146
6.3.3 Temperature dependence of the TEMPOL EAL rotational
correlation times and normalized component weights in frozen
aqueous solution with AmEtOH: 2% v/v DMSO . . . . . . . 149
6.3.4 Fluid/solid transition of the protein-associated domain and
in the mesodomain . . . . . . . . . . . . . . . . . . . . . . . . 151
6.3.5 Quantification of the composition and uniformity properties
of the PAD and mesodomain components . . . . . . . . 152
6.4 SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
7 Summary and Conclusion 157
7.1 State of the Art . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
7.2 Significance of the work presented in this dissertation . . . . . . . . 159
Appendix 163
C EPR spectrum from frozen aqueous solution at 0% DMSO, in the
absence of EAL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164
D The log�c and W values at different temperatures for the �EAL
with TEMPOL, 0 and 1% v/v DMSO system . . . . . . . . . . . . . 165
E Arrhenius parameters obtained from the rotational correlation time
for TEMPOL tumbling motion in the absence and presence of EAL,
0% and 1% v/v DMSO systems . . . . . . . . . . . . . . . . . . . . . 168
F Primers used in the development of the EAL mutants . . . . . . . . 169
G Reorientational correlation times and normalized weight values at
the different temperatures for EAL-4MT, 0 and 1% v/v DMSO . . . 170
H Enthalpy and entropy values for the equilibrium between Ws and
Wf mobility components in 1% v/v DMSO . . . . . . . . . . . . . . 172
I Reorientational correlation times and normalized
weight values at the different temperatures for EAL-4MT, 0.5, 2
and 4% v/v DMSO . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
J The log�c values at different temperatures for the EAL-AmEtOH
with TEMPOL, 0 and 2% v/v DMSO system . . . . . . . . . . . . . 176
K The log�c and W values at different temperatures for the �EAL
with TEMPOL, 0.5, 2, and 4% v/v DMSO system . . . . . . . . . . . 178
References 184
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