Dynamics and Cooperativity of the Catalytic Subunit of Protein Kinase AA Dissertation SUBMITTED

353 Pages·2015·11.59 MB·English

Dynamics and Cooperativity of the Catalytic Subunit of
Protein Kinase A

A Dissertation
SUBMITTED TO THE FACULTY OF THE
UNIVERSITY OF MINNESOTA

Jonggul John Kim

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF
DOCTOR OF PHILOSOPHY

Gianluigi Veglia

July 2015
© Jonggul John Kim 2015
ALL RIGHTS RESERVED
Pace non trovo, e non ho da far guerra;
E temo e spero, ed ardo e son un ghiaccio;
E volo sopra ‘l cielo e giaccio in terra;
E nullo stringo, e tutto il mondo abbraccio.

Tal m’ha in prigion, che non m’apre, né serra;
Né per suo mi riten, né scioglie il laccio;
E non m’ancide Amor, e non mi sferra;
Né mi vuol vivo, né mi trae d’imapaccio.

Veggio senz’ occhi; e non ho lingua e grido,
E bramo di perir, e cheggio aita;
Ed ho in odio me stesso ed amo altrui:

Pascomi di dolor, piangendo rido;
Equalmente mi spiace morte e vita:
In questo stato son, Donna, per Vui.
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Contents
Dedication ......................................................................................................................... i
List of Tables .................................................................................................................. vi
List of Figures ................................................................................................................ vii
List of Abbreviations ..................................................................................................... xi
0 Preface ................................................................................................................. 1
1 Overview of the catalytic subunit of the cAMP-dependent Protein Kinase
1.1 Architecture of PKA-C ........................................................................................... 5
1.2 Substrate Specificity of PKA-C .............................................................................. 7
1.3 Chemical Mechanism of PKA-C ............................................................................ 9
1.4 Major Conformational States of PKA-C during the Catalytic Cycle ..................... 14
1.5 Function of critical residues of PKA-C and insights into kinase function ............. 16
1.5.1 Small Lobe ............................................................................................... 16
1.5.2 Large Lobe .............................................................................................. 18
1.5.3 N and C-terminal Trails ............................................................................ 22
1.5.4 Hydrophobic Assembly of Kinase Core ................................................... 26
1.6 Dynamic nature of PKA-C ................................................................................... 28
2 Theory and Application of Solution NMR Spectroscopy
2.1 The Semi-classical Bloch model.......................................................................... 33
2.2 Density Matrix Approach ..................................................................................... 37
2.3 Hamiltonians of NMR .......................................................................................... 42
2.4 Coherence Transfer and Multi-dimensional NMR ............................................... 48
2.5 Nuclear Spin Relaxation ...................................................................................... 55
2.6 Cross-correlation and TROSY ............................................................................. 65
2.7 Cross-correlation in Methyl Groups ..................................................................... 69
2.8 Chemical Exchange: Study of molecular dynamics in the s-s timescale ........... 77
3 Isothermal Titration Calorimetry and Binding Cooperativity
ii
3.1 Isothermal Titration Calorimetry .......................................................................... 93
3.2 Cooperativity ....................................................................................................... 96
4 FLAMEnGO 2.0: an Enhanced Fuzzy Logic Algorithm for Structure-Based
Assignment of Methyl Group Resonances
4.1 Introduction ........................................................................................................ 103
4.2 Material and Methods ........................................................................................ 104
4.3 Results .............................................................................................................. 109
4.4 Discussion ......................................................................................................... 113
4.5 Conclusion ......................................................................................................... 116
4.6 Footnotes .......................................................................................................... 116
5 A Semi-Automated Assignment Protocol for Methyl Group Side-Chains in
Large Proteins
5.1 Introduction ........................................................................................................ 119
5.2 Labeling of side chain methyl groups for large proteins .................................... 120
5.3 Methyl labeling protocol for the cAMP-dependent Protein Kinase A ................. 121
5.4 Semi-Automated Methyl Group Resonance Assignment Strategies ................. 126
5.5 Semi-Automated Assignment Protocol using FLAMEnGO 2.0 .......................... 127
5.6 Conclusions and Perspectives .......................................................................... 133
5.7 Footnotes .......................................................................................................... 134
6 NMR Mapping of Protein Conformational Landscapes using Coordinated
Behavior of Chemical Shifts upon Ligand Binding
6.1 Introduction ........................................................................................................ 136
6.2 Methods ............................................................................................................. 138
6.3 Results .............................................................................................................. 142
6.4 Discussion ......................................................................................................... 147
6.5 Conclusion ......................................................................................................... 150
6.6 Footnotes .......................................................................................................... 150
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7 Synchronous Opening and Closing Motions are Essential for cAMP-
Dependent Protein Kinase A Signaling
7.1 Introduction ........................................................................................................ 152
7.2 Experimental Procedures .................................................................................. 154
7.3 Results .............................................................................................................. 156
7.4 Discussion ......................................................................................................... 162
7.5 Footnotes .......................................................................................................... 165
8 Mapping the Hydrogen Bond Networks in the Catalytic Subunit of Protein
Kinase A using H/D Fractionation Factors
8.1 Introduction ........................................................................................................ 167
8.2 Experimental Procedures .................................................................................. 169
8.3 Results .............................................................................................................. 171
8.4 Discussion ......................................................................................................... 175
8.5 Footnotes .......................................................................................................... 179
9 Dysfunctional Conformational Dynamics of Protein Kinase A Induced by a
Lethal Mutant of Phosphoslamban Hinder Phosphorylation
9.1 Introduction ........................................................................................................ 181
9.2 Materials and Methods ...................................................................................... 183
9.3 Results .............................................................................................................. 184
9.4 Discussion ......................................................................................................... 191
9.5 Footnotes .......................................................................................................... 195
10 Dysfunctional Conformational Dynamics of Protein Kinase A Induced by a
Lethal Mutant of Phosphoslamban Hinder Phosphorylation
10.1 Introduction ........................................................................................................ 197
10.2 Experimental Procedures .................................................................................. 199
10.3 Results .............................................................................................................. 202
10.4 Discussion ......................................................................................................... 207
10.5 Footnotes .......................................................................................................... 211
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Tables ........................................................................................................................... 213
Figures ......................................................................................................................... 220
References ................................................................................................................... 312

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List of Tables

4.1 Validation of static-based assignment of FLAMEnGO 2.0 ................................ 214
7.1 Summary of 15N relaxation and ITC data for PKA-CWT and PKA-CY204A ............ 215
7.2 Thermodynamics of ligand binding to PKA-CWT and PKA-CY204A ...................... 216
8.1 Average  values for the various structural motifs of PKA-C ............................ 217
10.1 The affinity, degree of cooperativity (), and thermodynamics of PKI binding
5-24
with respect to the saturated nucleotide .................................................................. 218
10.2 The affinity, degree of cooperativity (), and thermodynamics of PKI binding
5-24
with respect to the saturated ATP-competitive inhibitor ............................................ 219

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List of Figures
CHAPTER 1
1.1 Subdomains in PKA-C ...................................................................................... 221
1.2 Substrates and Inhibitor Peptide of PKA-C ....................................................... 222
1.3 Catalytic Mechanism of PKA-C ......................................................................... 223
1.4 Mg2+ binding sites in PKA-C .............................................................................. 224
1.5 Open, Intermediate, and Closed Conformational States ................................... 225
1.6 Pre- and post-phosphorylation Structures ......................................................... 226
1.7 Conserved Residues in the Small Lobe ............................................................ 227
1.8 Activation Loop Phosphorylation ....................................................................... 228
1.9 Interactions in the Large Lobe ........................................................................... 229
1.10 N-terminus of PKA-C ......................................................................................... 230
1.11 C-terminal Tail of PKA-C and Critical Residues ................................................ 231
1.12 Activation of Protein Kinases with Assembly of Hydrophobic Residues ............ 232
CHAPTER 2
2.1 Energy level of a I = ½ particle ......................................................................... 233
2.2 Schematic of a pulse and acquire experiment .................................................. 234
2.3 Single spin quantum operators ......................................................................... 235
2.4 In-phase and anti-phase single quantum operators ......................................... 236
2.5 Population Operators for a two spin system ..................................................... 237
2.6 Multiple quantum transitions and their operators .............................................. 238
2.7 Geometric anisotropy of dipolar coupling and chemical shift anisotropy .......... 239
2.8 Representation of evolution of two operators under a third operator ............... 240
2.9 Geometric representation of the product operator approach ............................ 241
2.10 Effect of the INEPT pulse sequence element ................................................... 242
2.11 HSQC pulse sequence and application to proteins .......................................... 243
2.12 Scheme and experiment for triple resonance experiments .............................. 244
2.13 Pulse sequence element the spin-state-selective coherence transfer ............. 245
2.14 Relaxation Mechanisms in NMR ...................................................................... 246
2.15 Physical models for interpretation of spectral density functions ....................... 247
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2.16 Spectral density functions for small and large molecules ................................. 248
2.17 Pulse sequences for 15N edited relaxation experiments ................................... 249
2.18 TROSY effect on a 1H-15N coupled spin system ............................................... 250
2.19 Pulse sequence and spectra of 1H-15N-TROSY HSQC ..................................... 251
2.20 Energy levels and spectral lines of an isolated methyl group ........................... 252
2.21 Dipolar fields for cross-correlation of 1H relaxation rates ................................. 253
2.22 Relaxation of multiple-quantum coherences in HMQC experiment .................. 254
2.23 Range of motions probed by NMR relaxation experiments .............................. 255
2.24 Chemical exchange on longitudinal magnetization .......................................... 256
2.25 Chemical exchange on transverse magnetization ............................................ 257
2.26 Effect of CPMG pulse train on chemical exchange .......................................... 258
2.27 Pulse sequence for a [1H-15N]-HSQC CPMG experiment ................................ 259
2.28 Pulse sequence for the TROSY Hahn-Echo experiment .................................. 260
CHAPTER 3
3.1 Schematic of ITC experiment ........................................................................... 261
3.2 Example ITC isotherms .................................................................................... 262
3.3 Thermodynamic cycles used to study ligand binding and mutation ................. 263
3.4 Mg2+ binding sites in PKA-C .............................................................................. 264
CHAPTER 4
4.1 GUI interface of FLAMEnGO 2.0 ....................................................................... 265
4.2 Application of FLAMEnGO 2.0 to PKA-C ......................................................... 266
4.3 The methyl-TROSY spectrum of PKA-C .......................................................... 267
4.4 Methyl Assignments confirmed with through-bond experiment ......................... 268

4.5 Distribution of mis-assigned methyl groups ...................................................... 269
CHAPTER 5
5.1 Biosynthetic pathways for methyl group labeling ............................................... 270
5.2 Growth of IVL labeled PKA-C ........................................................................... 271
5.3 SDS-PAGE gel of PKA-C ................................................................................. 272
5.4 13C-HMQC of Apo PKA-C ................................................................................. 273
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