Photolabeling of the Human Dopamine Transporter and theReactivity of Aryl Azides with Amino Acid Analogs Público
Holmes Morris, Muhsinah Lateefah (2008)
Abstract
This work explored the site of photolabeling of the human dopamine transporter (hDAT) by the photoaffinity agent [125I]-DEEP, an aryl azide photoaffinity label, and also the selectivity of aryl azides using phenyl azide and amino acid analogs. The site of incorporation of [125I]-DEEP had been shown to incorporate within transmembrane domains 1 and 2 of hDAT by Vaughan and Kuhar (1996). To further isolate the site of incorporation, [125I]-DEEP was photolyzed with hDAT membranes, isolated by SDS- PAGE, and digested with trypsin and cyanogen bromide (CNBr). The peptide, N93GGAFLVPYLLFM106, which is located at the top portion of TMD2, was identified as the possible site of [125I]-DEEP incorporation.
[125I]-DEEP contains a phenyl azide as the photoactive agent. In a followup study, the photolabeling selectivity of phenyl azide with amino acids was examined using analogs of amino acid side chains. Phenyl azide (100 μM) and amino acid analogs (1 M) were irradiated at 254 nm for 4 minutes in polar and nonpolar conditions and the kinetics of the reactions determined. 1-Octanethiol, butylamine, guanidine acetic acid, dimethyl sulfide, ethylbenzene, butyric acid, imidazole, and phenol were used to model the side- chains of cysteine, lysine, arginine, methionine, phenylalanine, glutamic acid, histidine, and tyrosine, respectively. N-ethylacetamide was used as a model for the peptide backbone.
The order of phenyl azide reactivity with analogs studied in polar conditions was guanidine acetic acid ≈ imidazole > phenol. The order of phenyl azide reactivity with analogs studied in nonpolar conditions was dimethyl sulfide > butyric acid ≈ butylamine >/= N-ethylacetamide ≈ 1-octanethiol ≈ ethylbenzene. Product stability in basic and acidic proteolytic conditions was also tested. Trypsin digestions were performed in 100mM ammonium bicarbonate at pH 8.9, while CNBr cleavages were performed in 70% trifluoroacetic acid at pH 1. Histidine, tyrosine and cysteine analogs were the most stable, while all of the other analogs were not as stable, and decomposed by greater than 50% within 24 hours. Product stability in proteolytic conditions has implications for photoaffinity labeling of proteins with aryl azides in which proteolysis is used for peptide mapping.
Table of Contents
Table of Contents
Page
Chapter One
Cocaine Addiction and the Human Dopamine Transporter 1
Cocaine Addiction 2
Available Treatments for Addiction 5
Cocaine as an Inhibitor of the Dopamine Transporter 6
Other Inhibitors to the Dopamine Transporter 10
Structure and Function of the Dopamine Transporter 13
Characterization of Membrane Bound Proteins 18
Photoaffinity Labeling as a Method to Identify the Cocaine
Binding Site of the Human Dopamine Transporter 25
Photoaffinity Labeling of DAT 31
Photochemistry of Aromatic Nitrenes 36
Project Overview and Objectives 39
Chapter Two
Photoaffinity Labeling of the Human Dopamine Transporter
with [125I]-DEEP51
Introduction52
Methods56
Cell Culture and Photoaffinity Labeling 57
Cell Culture 57
Membrane Preparation 57
Photoaffinity Labeling 58
Purification and Isolation of Radiolabeled hDAT 61
Immobilized Metal Affinity Chromatography 61
Gel Electrophoresis 61
Autoradiography 62
Western Blotting 62
Digestion of Radiolabeled hDAT 64
Enzymatic Digestions of Labeled hDAT 64
Chemical Cleavage 64
Thin Layer Chromatography 65
HPLC 66
SDS-PAGE of Extracted hDAT Peptides 67
Solid-State Photolabeling of hDAT with Radioligands 67
General Protein/Mass Analysis for Windows (GPMAW) 67
Results70
Introduction to the Results 71
Suitability of Photoaffinity Labeling Experimental Conditions 74
Investigations into Localizing the Cocaine Binging Site with the
GBR Analog, [125I]-DEEP 78
Solid-State Photoreactions of [125I]-DEEP and Amino Acids 89
Effect of Photoaffinity Labeling Conditions on [125I]-DEEP 92
Preliminary Investigations of [125I]-JJC 3-24, a Rimcazole
Photolabel of DAT 96
Discussion103
Introduction to the Discussion 104
Investigation of Photoincorporation Site of [125I]-DEEP on hDAT 105
Suitability of Photoaffinity Labeling Experiment Conditions 105
WIN 35, 428 Protection Experiment 106
Analysis of [125I]-DEEP Labeled Peptides Resulting from
Proteolytic Digestions 107
Primary CNBr Digestion followed by Secondary
Trypsin Digestion 107
Primary Trypsin Digestion followed by Secondary
CNBr Cleavage: Gel and TLC Analysis 112
HPLC of [125I]-DEEP Labeled hDAT 112
Solid-state Photolabeling of TM2 Amino Acids 113
Effect of Photoaffinity Labeling Conditions on [125I]-DEEP 114
Analysis of Photolabeling of hDAT with [125I]-JJC 3-24 117
Future Studies 118
Chapter Three
Photoreactions of Phenyl Azide with Amino Acid Analogs120
Introduction121
Methods124
Photoreactions of Phenyl Azide/Para-azidoacetophenone
with Amino Acid Analogs125
General Notes 125
Synthesis of Phenyl Azide 125
Purification of Phenyl Azide 126
Analysis of the Irradiation of Phenyl Azide
with Amino Acid Analogs127
Sample Preparation 127
High Pressure Liquid Chromatography Analysis 127
Irradiation of Phenyl Azide and Amino Acid Analogs
for HPLC and Mass Spectral Analysis 129
Irradiation of para-azidoacetophenone (PAAP) and
Amino Acid Analogs for HPLC and Mass Spectral Analysis 129
Irradiation of Phenyl Azide and Amino Acid Analogs
for 1H Nuclear Magnetic Resonance (NMR) 130
Collection of the Product of the Reaction between
Phenyl Azide and the Amino Acid Analogs 130
High Resolution Mass Spectrometry Analysis 130
Stability of Products 130
Stability of the Products of Phenyl Azide and
the Amino Acid Analogs at pH 8.9 in
Ammonium Bicarbonate 130
Stability of the Products of Phenyl Azide and
the Amino Acid Analogs at 25oC in
Trifluoroacetic Acid (TFA), pH 1 131
Data Analysis 1 31
Results136
Introduction to the Results137
Photoreactions with Phenyl Azide and Butylamine as a Model for Lysine139
Phenyl Azide Photoreactivity with Butylamine as a Model for Lysine 139
Phenyl Azide Photoreactivity with Butylamine as a Model for Lysine
under NEAT conditions 148
The Identification of N-butyl-2H-azepin-7-amine by Electrospray
Ionization (ESI) Mass Spectrometry (MS) and Proton Nuclear
Magnetic Resonance (1H NMR) 154
Product Stability of Butylazepine under Proteolytic Conditions 159
Photoreactions with Phenyl Azide and 1-Octanethiol as a Model for Cysteine162
Phenyl Azide Photoreactivity with 1-Octanethiol as a Model for Cysteine 162
Phenyl Azide Photoreactivity with 1-Octanethiol as a Model for Cysteine
in NEAT Conditions 171
The Identification of 7-(octylthio)-2H-azepine by Atmospheric Pressure
Chemical Ionization (APCI) Mass Spectrometry (MS) 177
Product Stability of Octylthioazepine under Proteolytic Conditions 181
Single Exponential First-Order Rate Constants for Phenyl Azide
Irradiated with Other Amino Acid Analogs 184
Dimerization of Phenyl Azide195
Double Exponential Rate Constants Applied to the Photoreactions of Phenyl
Azide with Amino Acid Analogs203
Identification of Products by Mass Spectrometry for Aryl Azides Irradiated
with Amino Acid Analogs213
Products from the Irradiation of Phenyl Azide with Amino Acid Analogs218
Stability of Products in Proteolytic Conditions223
Discussion226
Introduction to the Discussion227
Reactivity of Phenyl Azide with Amino Acid Analogs
Interpretation of Results for Photoreactions of Phenyl Azide
with Amino Acid Analogs, Butylamine and 1-Octanethiol 229
Butylamine as a Model for Lysine 229
1-Octanethiol as a Model for Cysteine 230
Interpretation of Results for Photoreactions of Phenyl Azide with Amino
Acid Analogs in Nonpolar, Aqueous, and NEAT Conditions 233
Nonpolar Conditions 233
Aqueous Conditions 234
NEAT Conditions 234
Phenyl Azide Irradiated in Nonpolar and Aqueous Conditons 238
Interpretation of Phenyl Azide Reactivity with Amino Acid
Analogs 240
Discussion of the Product Formation of Azepines from the Irradiation
of Aryl Azides and Amino Acid Analogs245
Identification of Butylazepine
Analysis of Azepine Products from Phenyl Azide and Amino Acid
Analogs using Mass Spectrometry 247
Absorbance of Products from Photoreactions of Phenyl Azide and Amino
Acid Analogs248
Azepine Product Stability in Proteolytic Conditions252
Stability of Product in Basic Conditions 252
Stability of Product in Acidic Conditions 253
Correlation of Rate Constants vs. Product Stability in Proteolytic
Conditions 258
So What Gets Labeled? 260
References262
Appendices299
List of Figures
Chapter One
Cocaine Addiction and the Human Dopamine Transporter1
Figure 1-1. Forms of Cocaine 3
Figure 1-2. Dopaminergic Neurotransmission 8
Figure 1-3. DAT and VMAT Topology 9
Figure 1-4. DAT Inhibitors 12
Figure 1-5. 2-D Topology of the Human Dopamine Transporter (hDAT) 16
Figure 1-6. Proposed Mechanism for the Glutamate Transporter 20
Figure 1-7. The LeuTAa Topology 23
Figure 1-8. Structure of the LeuT-desipramine Complex 24
Figure 1-9. Steps for Photoaffinity Labeling 27
Figure 1-10. Typical Compounds used as Photoaffinity Labels 28
Figure 1-11. Photolysis of Photophores 29
Figure 1-12. GBR Analogs 34
Figure 1-13. Regions of Incorporation of PALs on hDAT 35
Figure 1-14. Pathways for Photolysis of Phenyl Azide 38
Figure 1-15. Structures of Cocaine and DAT Photolabels 41
Figure 1-16. Analogs of DAT Photolabels 42
Figure 1-17. Intermediates of Singlet and Triplet Nitrenes 49
Chapter Two
Photoaffinity Labeling of the Human Dopamine Transporter
with [125I]-DEEP 51
Figure 2-1. Structures of Cocaine and hDAT Inibitors 53
Figure 2-2. [125I]-DEEP as an Example of a DAT Photolabel 54
Figure 2-3. Western Blot of Purified DAT 75
Figure 2-4. [125I]-RTI-82 Labels Near the Cocaine Binding Site 76
Figure 2-5. Time-Dependent Irradiations with [125I]-RTI-82 77
Figure 2-6. [125I]-DEEP Labels Near the Cocaine Binding Site 80
Figure 2-7. Primary Proteolytic Digestions of [125I]-DEEP Labeled hDAT 81
Figure 2-8. HPLC of Primary Tryptic Digestion of [125I]-DEEP hDAT 82
Figure 2-9. TLC of Secondary Digestions of [125I]-DEEP hDAT 83
Figure 2-10. HPLC Analysis of Secondary CNBr Digestion of Tryptic Peptide,
T1 84
Figure 2-11. HPLC Analysis of Secondary CNBr Digestion of Tryptic Peptide,
T2 85
Figure 2-12. Primary Digestion of [125I]-DEEP Labeled hDAT with Trypsin
TPCK 87
Figure 2-13. HPLC Radioactive Trace of [125I]-DEEP with Methionine 89
Figure 2-14. HPLC Radioactive Trace of [125I]-DEEP with Leucine 90
Figure 2-15. HPLC Radioactive Trace of [125I]-DEEP with Tyrosine 91
Figure 2-16. Effect of Irradiation on [125I]-DEEP 92
Figure 2-17. Effect of Tryptic Conditions on [125I]-DEEP Labeled hDAT 93
Figure 2-18. Effect of CNBr/TFA Solution on the Photoaffinity Label,
[125I]-DEEP 94
Figure 2-19. TFA causes Decomposition of [125I]-DEEP Photolabel 95
Figure 2-20. High Concentration of [125I]-JJC 3-24 Labels hDAT 97
Figure 2-21. SDS-PAGE of Primary Tryptic Digestion of hDAT Labeled with
[125I]-DEEP and [125I]-JJC 3-24 98
Figure 2-22. Primary CNBr Cleavage of [125I]-JJC 3-24 Labeled hDAT 99
Figure 2-23. [125I]-JJC 3-24 Labels hDAT Peptide, PLFYM 100
Figure 2-24. Effect of Irradiation on [125I]-JJC 3-24 101
Figure 2-25. Effect of CNBr Conditions on [125I]-JJC 3-24 102
Figure 2-26. CNBr Cleavage Sites of hDAT 109
Figure 2-27. Primary CNBr and Secondary Tryptic Digestion Cleavage Sites 110
Figure 2-28. Primary Tryptic and Secondary CNBr Digestion Cleavage Sites 111
Figure 2-29. Effect of [125I]-DEEP on the Retention Time of Amino Acids 116
Chapter Three
Photoreactions of Phenyl Azide with Amino Acid Analogs120
Figure 3-1. Phenyl Azide and Butylamine in Cyclohexane Before Irradiation 140
Figure 3-2. Phenyl Azide and Butylamine in Cyclohexane After Irradiation 141
Figure 3-3. Phenyl Azide and Butylamine in Cyclohexane Before and After
Irradiation 142
Figure 3-4. Phenyl Azide and Butylamine in Cyclohexane Irradiated over Time 143
Figure 3-5. Single Exponential First-Order Decay Curve Fit for Phenyl Azide
and Butylamine in Cyclohexane 144
Figure 3-6. Double Exponential First-Order Decay Curve Fit for
Phenyl Azide and Butylamine in Cyclohexane 145
Figure 3-7. Phenyl Azide and Butylamine in Cyclohexane Product Formation 146
Figure 3-8. Phenyl Azide and Butylamine (NEAT) Before and After Irradiation 149
Figure 3-9. Phenyl Azide and Butylamine (NEAT) Irradiated over Time 150
Figure 3-10. Single First Order Exponential Decay Curve Fit for Phenyl Azide
and Butylamine (NEAT) 151
Figure 3-11. Phenyl Azide and Butylamine (NEAT) Product Formation 152
Figure 3-12. Mass Spectrometry of the Butylazepine Product 156
Figure 3-13. Experimental Proton NMR of N-butyl-3H-azepin-2-amine. 157
Figure 3-14. Theoretical Proton NMR of N-butyl-3H-azepin-2-amine 158
Figure 3-15. Stability of Butylazepine Product in Basic Conditions 160
Figure 3-16. Stability of Butylazepine in Acidic Conditions 161
Figure 3-17. Phenyl Azide and 1-Octanethiol in Cyclohexane Before Irradiation 163
Figure 3-18. Phenyl Azide and 1-Octanethiol in Cyclohexane After Irradiation 164
Figure 3-19. Phenyl Azide and 1-Octanethiol in Cyclohexane Before and After Irradiation 165
Figure 3-20. Phenyl Azide and 1-Octanethiol in Cyclohexane Irradiated
over Twenty Minutes 166
Figure 3-21. Single Exponential First-Order Decay Curve Fit for Phenyl Azide
and 1-Octanethiol in Cyclohexane 167
Figure 3-22. Double Exponential Decay Curve Fit for Phenyl Azide and
1-Octanethiol in Cyclohexane 169
Figure 3-23. Product Formation for Phenyl Azide and 1-Octanethiol
in Cyclohexane 170
Figure 3-24. Phenyl Azide and 1-Octanethiol (NEAT) Before and After
Irradiation 172
Figure 3-25. Phenyl Azide and 1-Octanethiol (NEAT) Irradiated over
Sixty Minutes 173
Figure 3-26. Phenyl Azide and 1-Octanethiol (NEAT) First-Order
Exponential Decay Curve Fit 174
Figure 3-27. Rate of Product Formation for Phenyl Azide and
1-Octanethiol (NEAT) 176
Figure 3-28. Mass Spectrometry of the Octylthioazepine Product 178
Figure 3-29. Stability of Octylthioazepine in Basic Conditions 182
Figure 3-30. Stability of Octylthioazepine in Acidic Conditions 183
Figure 3-31. Single Exponential First-Order Curve Fit for Phenyl Azide
and Imidazole 185
Figure 3-32. Single Exponential First-Order Curve Fit for Phenyl Azide
and Guanidine Acetic Acid 186
Figure 3-33. Single Exponential First-Order Curve Fit for Phenyl Azide
and Phenol 187
Figure 3-34. Single Exponential First-Order Curve Fit for Phenyl Azide
and N-ethylacetamide 188
Figure 3-35. Single Exponential First-Order Curve Fit for Phenyl Azide
and N-ethylacetamide (NEAT) 189
Figure 3-36. Single Exponential First-Order Curve Fit for Phenyl Azide
and Ethylbenzene 190
Figure 3-37. Single Exponential First-Order Curve Fit for Phenyl Azide
and Butyric Acid 191
Figure 3-38. Single Exponential First-Order Curve Fit for Phenyl Azide
and Dimethyl Sulfide First-Order Curve Fit 192
Figure 3-39. Loss of Phenyl Azide in Aqueous Conditions for 4 Minutes
of Irradiation 197
Figure 3-40. Dimerization of Phenyl Azide in Aqueous Conditions 198
Figure 3-41. Loss of Phenyl Azide in Cyclohexane for 4 Minutes of Irradiation 199
Figure 3-42. Dimerization of Phenyl Azide in Cyclohexane 200
Figure 3-43. Double Exponential First-Order Curve Fit for Phenyl Azide
and Guanidine Acetic Acid 204
Figure 3-44. Double Exponential First-Order Curve Fit for Phenyl Azide
and Imidazole 205
Figure 3-45. Double Exponential First-Order Curve Fit for Phenyl Azide
and Phenol 206
Figure 3-46. Double Exponential First-Order Curve Fit for Phenyl Azide
and Dimethyl Sulfide 208
Figure 3-47. Double Exponential First-Order Curve Fit for Phenyl Azide
and Butyric Acid 209
Figure 3-48. Double Exponential First-Order Curve Fit for Phenyl Azide
and N-ethylacetamide 210
Figure 3-49. Double Exponential First-Order Curve Fit for Phenyl Azide
and Ethylbenzene 211
Figure 3-50. Product Peak Heights for Cysteine, Lysine, and Peptide
Backbone Analogs 219
Figure 3-51. Product Peak Heights in Nonpolar Environment 220
Figure 3-52. Product Peak Heights in Aqueous Conditions 221
Figure 3-53. Product Peak Heights in NEAT Conditions 222
Figure 3-54. Stability of Products in Basic Proteolytic Conditions 224
Figure 3-55. Stability of Products in Acidic Proteolytic Conditions 225
Figure 3-56. UV Absorbance of Products in Different Solvent Conditions 250
Figure 3-57. Acid Hydrolysis of Azepine 259
Figure 3-58. Azepine becomes the Protonated Salt upon Addition of Acid 259
List of Tables
Chapter One
Cocaine Addiction and the Human Dopamine Transporter1
Table 1-1. Amino Acid Analogs 44
Chapter Two
Photoaffinity Labeling of the Human Dopamine Transporter
with [125I]-DEEP 51
Table 2-1. HPLC Gradient Description of Sara3 Method 66
Table 2-2. GPMAW Codes for Theoretical Proteolytic Digestions 68
Table 2-3. Summary of Peptides from Primary and Secondary Digestions of
[125I]-DEEP Labeled hDAT 86
Chapter Three
Photoreactions of Phenyl Azide with Amino Acid Analogs120
Table 3-1. HPLC Gradient Description for Method SHORT1 128
Table 3-2. HPLC Gradient Description for Method 16 128
Table 3-3. Single Exponential First-Order Rate Constants 193
Table 3-4. Single Exponential First-Order Rate Constants Divided
by Solvent Conditions 194
Table 3-5. Initial Reaction Rates of Phenyl Azide Reacting with
Amino Acid Analogs 201
Table 3-6. Single and Double Exponential Rate Constants for Amino
Acid Analogs in Aqueous Conditions 207
Table 3-7. Single and Double Exponential Rate Constants for Amino
Acid Analogs in Cyclohexane 212
Table 3-8. Mass Spectrometry Analysis of Amino Acid Analogs with
Phenyl Azide 214
Table 3-9. Mass Spectrometry Analysis of Amino Acid Analogs with
Para-azidoacetophenone (PAAP) 216
Table 3-10. Rate Constants of Amino Acids in Nonpolar Conditions 235
Table 3-11. Rate Constants of Amino Acids in Aqueous Conditions 237
Table 3-12. Partial List of Amino Acids Labeled by Azido Photolabels 242
Table 3-13. Arylnitrene Reactivity with Amino Acid Functional Groups 244
Table 3-14. Product Stability in Basic and Acidic Conditions 255
List of Schemes
Chapter One
Cocaine Addiction and the Human Dopamine Transporter1
Scheme 1-1. Photolysis of Phenyl Azide 48
Chapter Two
Photoaffinity Labeling of the Human Dopamine Transporter
with [125I]-DEEP 51
Scheme 2-1. Localization of Photoaffinity Labels 60
Chapter Three
Photoreactions of Phenyl Azide with Amino Acid Analogs120
Scheme 3-1. Irradiation of Phenyl Azide forms 3H-Azepine and Azobenzene 123
Scheme 3-2. Reaction Schemes for Butylamine and Phenyl Azide 154
Scheme 3-3. Reaction Scheme for 1-Octanethiol and Phenyl Azide 177
Scheme 3-4. Photolysis of Phenyl Azide in Aqueous and Organic Solvents 246
Scheme 3-5. Photoisomerization of 3H-Azepines 257
Scheme 3-6. Irradiation of N-butyl-3H-azepin-2-amine 257
List of Appendices
A. The Amino Acid Sequence of the Flag 6XHis Tagged Human
Dopamine Transporter299
B. Correlations of Physical Properties vs. Rate Constants302
Table of Physical Constants for the Amino Acid Analog
Functional Groups 302
Correlation Between Phenyl Azide Reactivity and Electronegativity 303
Correlation Between the Phenyl Azide Reactivity and Dielectric Constant 304
Correlation Between the Phenyl Azide Reactivity and Polarizability 305
C. ANOVA Data Tables for the Rate Constants306
One-Way ANOVA of Rate Constants in Cyclohexane 306
One-Way ANOVA of Rate Constants in Aqueous Conditions 308
One-Way ANOVA of Rate Constants in NEAT Conditions 309
D. Correlations of Rate Constants vs. Product Stability in
Proteolytic Conditions310
Correlation Between the Phenyl Azide Reactivity and Product Stability
in Acidic Conditions 310
Correlation Between the Phenyl Azide Reactivity and Product Stability
in Basic Conditions 311
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