Table Of ContentRAMAN SPECTROSCOPIC IMAGING ANALYSIS OF SIGNALING PROTEINS AND
PROTEIN COFACTORS IN LIVING CELLS
Achut P Silwal
A Dissertation
Submitted to the Graduate College of Bowling Green
State University in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
August 2018
Committee:
H Peter Lu, Adviser
Anita Simic
Graduate Faculty Representative
John R Cable
Alexey T Zayak
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ABSTRACT
H Peter Lu, Advisor,
Proteins play a central role in biological processes. Broad classes of protein types are
involved in the innumerable functions of living systems, such as catalysis of the biochemical
reactions, transportation of the essential molecules, defense of the immune system, and the
transmission of messages from cells to cells. In our projects, we have combined the analytical
approaches including surface-enhanced Raman scattering (SERS), fluorescence microscopy,
electrochemistry, and computational methods to investigate the structures and functions of the
proteins and signaling molecules in the living cells. In our research, we have studied the
interactions and functions of dopamine transporters (DAT), dopamine receptors (DARs), and
several signaling molecules such as dopamine (DA), amphetamine (AMP), methamphetamine
(MAMP), and methylenedioxypyrovalerone (MDPV) in living cells. The interactions between
signaling molecules and DAT or DARs are crucial for the functioning of dopaminergic
pathways. In our project, we have probed interactions of signaling proteins including DAT and
DARs and investigated the changes that happen in signaling proteins, other interacting
compounds or intracellular contents of the second messenger such as cyclic adenosine
monophosphate (cAMP). The second messenger like cAMP is important in many biological
processes which are produced due to interactions of the signaling protein in living cells. Our
studies on DA-DAT or DA-DARs interactions mainly utilize the Raman spectroscopy to
characterize the selectivity and efficacy of psychopharmaceutic drugs. In addition, we have also
studied the redox states and mechanism of protein cofactors in different experimental conditions.
We have probed and characterized the redox states and mechanisms of FMN cofactors in
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biological and non-biological environments using electrochemistry, SERS measurements, and
computational measurements. The results obtained from our research could be useful in the
diagnosis of abnormalities or diseases originated from malfunctioning of proteins or protein-
ligand interactions.
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To my friends and family
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ACKNOWLEDGMENTS
Firstly, I would like to express my deepest gratitude to my advisor Prof. Dr. H. Peter Lu
for being a caring person and constant source of support, ideas, and inspiration throughout my
Ph.D. research.
Besides my advisor, I would like to thank my committee members: Dr. John R. Cable,
Dr. Alexey T. Zayak, and Dr. Anita Simic for their valuable time, insightful comments, and
encouragement.
I would also like to thank Dr. Jon E. Sprague, Director of the Ohio Attorney General’s
Center for the Future of Forensic Science at BGSU for his help and collaboration. I would also
like to thank Dr. Andrew Torreli for the stimulating discussion on protein biology.
My sincere thanks go to present and past members of my research group; especially Dr.
Yufan He and Dr. Takashige Fujiwara for their helpfulness and teaching. I would also like to
thank Dr. Bharat Dhital, Dr. Dibeyndu K. Sasmal, Dr. Rajeev Yadav, Dr. Nibedita Pal, Dr.
Vishal Govind Rao, Dr. Zijian Wang, Dr. Qing Guo, Dr. Maolin Lu, and Min Gu for all research
techniques and ideas they taught me.
I am grateful to my fellow friends Meiling Wu, Susovan Roy Chowdhury, Sunidhi
Jaiswal, and Lorena Alvarez for their friendship, supports, and stimulating discussions.
I am thankful to the Bowling Green State University (BGSU); Department of Chemistry
and Center for Photochemical Sciences; the Ohio Eminent Scholar endowment fund, and the
Ohio Attorney General’s center for the future of Forensic Science for funding.
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I am thankful to all my professors at BGSU for providing me the best education. I am
also thankful to staffs at the Center for Photochemical Sciences and the Department of Chemistry
particularly Nora R. Cassidy, Alita Frater, Hilda E. Miranda, Mary Toth, Charles Codding, and
Doug Martin for their kind help.
Last but not the least, I would like to thank my family: my parents and siblings for their
love and support. I am very thankful to my beloved wife, Rakshya Khatiwada, for being such a
wonderful person and supporting me all the time with love and understanding. I am thankful to
all who helped me unconditionally to make this work possible and enjoyable.
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TABLE OF CONTENTS
Page
CHAPTER 1. INTRODUCTION ......................................................................................... 1
1.1. Biological significances of proteins .................................................................... 1
1.2 Signaling proteins ................................................................................................ 2
1.3. Dopamine in the central nervous system (CNS) ................................................. 3
1.3.1 Mechanism of dopaminergic signaling ................................................. 5
1.3.2 Dopamine transporter ............................................................................ 7
1.3.3 Dysfunction of dopamine transporter and neurodegenerative diseases 11
1.3.4 Dopamine receptors .............................................................................. 11
1.4 Flavoproteins ........................................................................................................ 13
1.4.1 Flavin cofactors ..................................................................................... 15
1.4.2 Electron transfer process in Flavin coenzymes ..................................... 16
1.5 Raman spectroscopy ............................................................................................ 18
1.5.1 Surface-enhanced Raman spectroscopy (SERS) .................................. 19
1.5.2 Electromagnetic mechanism ................................................................. 20
1.5.3 Chemical enhancement mechanism ...................................................... 24
1.6 References ............................................................................................................ 25
CHAPTER 2. EXPERIMENTAL SECTION ........................................................................ 43
2.1 Theoretical background ....................................................................................... 43
2.1.1 Raman spectroscopy ............................................................................. 43
2.1.2 Resonance Raman spectroscopy ........................................................... 45
2.1.3 Surface-enhanced Raman spectroscopy ................................................ 46
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2.1.4 Two-photon excited (2PE) fluorescence microscopy ........................... 48
2.1.5 Transmission electron microscopy (TEM) ........................................... 50
2.1.6 Confocal microscopy ............................................................................ 53
2.2 Experimental set-ups ............................................................................................ 54
2.2.1 Raman measurements and optical imaging ........................................... 54
2.2.2 Electrochemical control and Raman measurement ............................... 55
2.2.3 Two-photon excitation (2PE) fluorescence imaging ............................ 57
2.2.4 Two-photon excited (2PE) fluorescence microscopy ........................... 58
2.3 Density functional theory calculations ................................................................. 59
2.4 Materials and sample preparation ........................................................................ 60
2.4.1 Synthesis of silver nanoparticles ........................................................... 60
2.4.2 Fabrication of silica shell on silver NP ................................................. 61
2.4.3 Preparation of Britton-Robinson (B-R) buffer ...................................... 62
2.4.4 HEK293 cell culture ............................................................................. 62
2.4.5 Preparation of hDAT inducible HEK293 cells ..................................... 62
2.4.6 Bacteria growth and Plasmid amplification .......................................... 63
2.4.7 Transfection of pcDNA3.1-hDAT in HEK293 cell .............................. 64
2.5 References ............................................................................................................ 65
CHAPTER 3. RAMAN SPECTROSCOPIC SIGNATURE MARKER OF DOPAMINE-
HUMAN DOPAMINE TRANSPORTER INTERACTION IN LIVING CELLS ................ 75
3.1 Introduction .......................................................................................................... 76
3.2 Experimental sections .......................................................................................... 83
3.2.1 Synthesis of silver nanoparticles and sample preparation .................... 83
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3.2.2 Surface-enhanced Raman measurements .............................................. 84
3.2.3 Two-dimensional SERS plot vs relative signal peak intensity ............. 84
3.2.4 Density functional theory calculations .................................................. 85
3.3 Results and discussion ......................................................................................... 85
3.4 Summary ............................................................................................................ 95
3.5 References ........................................................................................................... 96
CHAPTER 4. MODE-SELECTIVE RAMAN IMAGING OF DOPAMINE-HUMAN
DOPAMINE INTERACTION IN LIVE CELLS .................................................................. 109
4.1 Introduction .......................................................................................................... 110
4.2 Experimental sections .......................................................................................... 114
4.2.1 Synthesis of silica-coated silver nanoparticles ..................................... 114
4.2.2 HEK293 cells culture ............................................................................ 115
4.2.3 Preparation of hDAT inducible HEK293 cells ..................................... 115
4.2.4 Transfection of pcDNA3.1-hDAT in HEK293 cell .............................. 116
4.2.5 Two photons excited (2PE) fluorescence imaging ............................... 116
4.2.6. Surface-enhanced Raman measurements ............................................. 116
4.2.7 Mode-selective Raman measurement ................................................... 117
4.2.8 Density functional theory calculations .................................................. 117
4.3 Results and discussion ......................................................................................... 118
4.3.1 Conventional and mode-selective Raman measurement ...................... 118
4.3.2 Two-photon excited (2PE) fluorescence imaging approach ................. 124
4.3.3 Correlation between mode-selective Raman image and spectra ........... 128
4.4 Summary ............................................................................................................ 130
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4.5 References ............................................................................................................ 131
CHAPTER 5. RAMAN SPECTROSCOPIC ANALYSIS OF SIGNALING MOLECULES-
DOPAMINE RECEPTORS INTERACTIONS IN LIVING CELLS ................................... 139
5.1 Introduction .......................................................................................................... 140
5.2 Experimental section ............................................................................................ 145
5.2.1 Transfection of DRD1 and DRD2 DNA in live cells ........................... 145
5.2.2 Synthesis of silica-coated silver nanoparticles ..................................... 147
5.2.3 Density functional theory calculations .................................................. 147
5.3 Results and discussion ......................................................................................... 148
5.3.1 Internalization of silver nanoparticles ................................................... 148
5.3.2 Probing of intracellular cAMP in HEK293 cells .................................. 150
5.3.3 Probing of intracellular cAMP in HT22 cells ....................................... 153
5.3.4 Raman peaks assignment of cAMP ...................................................... 160
5.3.5 Raman peaks assignment of DRD1-HEK293 cells .............................. 165
5.4 Summary ............................................................................................................ 166
5.5 References ............................................................................................................ 167
CHAPTER 6. RAMAN SPECTROSCOPY PROBING OF REDOX STATES AND
MECHANISM OF FLAVIN COENZYME .......................................................................... 179
6.1 Introduction .......................................................................................................... 179
6.2 Experimental section ............................................................................................ 189
6.2.1 Synthesis of silver nanoparticles and sample preparation .................... 189
6.2.2 Density functional theory calculations .................................................. 189
6.2.3 Surface-enhanced Raman measurements and electrochemical control 190
