Chemically Induced Dynamic Nuclear Polarization of 19F Nuclei A thesis submitted for the degree of Doctor of Philosophy by Ilya Kuprov Trinity term 2005 Corpus Christi College Oxford University Chemically Induced Dynamic Nuclear Polarization of 19F Nuclei A thesis submitted for the degree of Doctor of Philosophy by Ilya Kuprov Corpus Christi College Trinity term 2005 This study explores, both theoretically and experimentally, the photochemically induced dynamic nuclear polarization (photo-CIDNP) of 19F nuclei, the associated spin relaxation, cross-relaxation and cross-correlation effects, as well as potential applications of 19F CIDNP to protein structure and folding problems. It was demonstrated that in the flavin mononucleotide / 3-fluorotyrosine system and in the whole class of structurally related photochemical systems the 19F spin polarization exceeds the Boltzmann level by nearly two orders of magnitude and may be sustained in this elevated state indefinitely by continuous laser irradiation of the sample. A theoretical model describing photo-CIDNP magnetization evolution in a continuously photochemically pumped fluorine-proton nuclear system was assembled and tested. It was found that an accurate account of DD-CSA cross-correlation is essential for the correct description of the temporal behaviour and relaxation of the chemically pumped 13C, 15N and 19F nuclear magnetic systems. The sign and the amplitude of the observed 19F CIDNP effect were found to depend strongly on the effective rotational diffusion correlation time, giving an in situ probe of this parameter for these rapidly evolving systems. The origin of this effect has been traced to electron- nuclear cross-relaxation effects in the intermediate radicals. To counteract light intensity decay in optically dense samples, an efficient and inexpensive method for in situ laser illumination of NMR samples inside the narrow superconducting magnet bore has been developed. It utilises a stepwise tapered optical fibre to deliver light uniformly along the axis of a 5 mm NMR tube. Application of the theoretical and experimental framework described above to the uniformly fluorotyrosinated Green Fluorescent Protein and Trp-cage protein allowed the characterization of the protein surface structure in the immediate vicinity of the fluorotyrosine residues, and provided information on the motional regimes of all the labelled residues. The results demonstrate the power and versatility of CIDNP based structure investigation methods, particularly for mapping out the solvent-accessible amino acid residues and their surroundings and giving an order of magnitude sensitivity increase in 19F nuclear magnetic resonance experiments. Acknowledgements I would like to thank Professor Peter Hore for helping me to escape from the ruins of Soviet Union, for the very encouraging and wise supervision, and for continuing support and lots of good advice. I am grateful to Professor Martin Goez, who shared with me his vast practical experience with flash-photolysis and NMR hardware. Many thanks to Prof. Valeri Kopytov, Prof. Margarita Kopytova, Prof. Viktor Bagryansky, Prof. Brian Howard, Prof. Peter Atkins, Prof. Nick Trefethen from Oxford and Novosibirsk, whose excellent lectures have formed the core of what I presently know and a point of reference for my own teaching endeavours. I am indebted to Prof. Ludmila Krylova, Dr. Anatoly Golovin and Dr. Viktor Mamatyuk, for the wise supervision and providing the much needed spectrometer time in the early days. I thank the Oxford Supercomputing Centre for providing about 2 years’ worth of CPU time, Nick Soffe for help with the spectrometer hardware, Pete Biggs and David Temple for keeping the IT infrastructure running, Tim Craggs, Farid Khan and Sophie Jackson for preparing the fluorinated GFP, Prof. Niels Andersen for cooking the fluorinated Trp- cage protein. Loud cheers go to the jolly and diverse PJH and CRT group crowd. To Nicola Wagner and Chris Rodgers for always being there to share a laugh. To Iain Day for showing me around and helping to get started. To Ken Hun Mok for very helpful discussions, for volunteering to verify my 15N and 19F relaxation theory calculations, and jointly with Alexandra Yurkovskaya, for showing that science is not all roses. Many thanks to Corpus Christi College for being my home for the last three years. The financial support from Scatcherd Europeran Foundation and Hill Foundation is gratefully acknowledged with special thanks to Anthony Smith, Alastair Tulloch and Susan Richards of Hill Foundation for always being there to help. Special thanks to Olga for making the world a much better place to be. Publications arising directly from work described in this thesis 1. Chemically amplified 19F-1H nuclear Overhauser effects I. Kuprov, P.J. Hore, J. Magn. Reson. 168, 1-7 (2004). 2. Uniform illumination of optically dense NMR samples I. Kuprov, P.J. Hore, J. Magn. Reson. 171, 171-175 (2004). 3. Design and performance of a microsecond time-resolved photo-CIDNP add-on for a high-field NMR spectrometer I. Kuprov, M. Goez, P.A. Abbott, P.J. Hore, Rev. Sci. Instrum. 77, 084103 (2005) 4. Increasing the sensitivity of time-resolved photo-CIDNP experiments by multiple laser flashes and temporary storage in the rotating frame M. Goez, I. Kuprov, P.J. Hore, J. Magn. Reson. 177, 146-152 (2005) Publications related to the work described in this thesis 1. Bidirectional electron transfer in Photosystem I: determination of two distances + − between P700 and A1 in spin-correlated radical pairs S. Santabarbara, I. Kuprov, W.V. Fairclough, P.J. Hore, S. Purton, P. Heathcote, M.C.W. Evans, Biochemistry, 44, 119-2128 (2005). Contents 1 Theoretical framework 1 1.1 Brief review of spin relaxation in non-viscous liquids 1 1.1.1 General formalism 1 1.1.2 Relaxation and cross-relaxation caused by magnetic dipole interaction 4 1.1.3 Relaxation caused by rotational modulation of the Zeeman interaction 10 1.1.4 Cross-correlated relaxation 11 1.2 Origins of the photo-CIDNP effect 13 1.3 Secondary radical kinetics in the photo-CIDNP generation 17 2 19F nucleus: exploratory calculations and photo-CIDNP experiments 23 2.1 Introduction 23 2.2 Experimental details 25 2.3 Computational detail 27 2.4 Ab initio hyperfine coupling constants 28 2.4.1 General notes 28 2.4.2 The role of the solvent interactions 29 2.4.3 Tight basis functions 30 2.4.4 Sharp hyperfine couplings 31 2.4.5 Fluorophenoxyl radicals 32 2.4.6 Fluorotyrosyl radicals 35 2.4.7 Fluorotryptophan radicals 36 2.4.8 Rotational dependence of the hyperfine couplings 39 2.5 Experimental reconaissance 42 2.5.1 2-fluorophenol 43 2.5.2 3-fluorophenol 46 2.5.3 4-fluorophenol 48 2.5.4 Fluorotryptophans 49 2.5.5 Fluorotyrosines 51 2.6 Summary 53 3 Chemically pumped 19F-1H cross-relaxation 55 3.1 Introduction 55 3.2 Chemically pumped 19F-1H cross-relaxation – theoretical setup 56 3.3 Experimental methods 58 3.4 Computational methods 59 3.5 Results 59 3.6 Discussion 63 4 Uniform illumination of optically dense NMR samples 67 4.1 Introduction 67 4.2 Materials and methods 69 4.3 Results and discussion 70 4.4 Prolonged sample illumination: ways and consequences 74 4.5 Square-cut vs. tapered illuminating tip: performance benchmarks 77 4.6 Concluding remarks 79 5 Fluorotyrosine-labelled Trp-cage protein 81 5.1 Introduction 81 5.2 Solid phase peptide synthesis 82 5.3 (N)-FMOC-(O)-tBu-3-fluoro-L-tyrosine synthesis 86 5.4 Results and discussion 88 6 19F NMR and photo-CIDNP study of green fluorescent protein 95 6.1 Introduction 95 6.2 Materials and methods 98 6.3 Theoretical framework 99 6.4 Results and discussion 101 6.4.1 19F signal assignment and NMR data 101 6.4.2 Ring-flip isomers 102 6.4.3 Relaxation analysis 103 6.4.4 Photo-CIDNP experiments 104 6.4.5 Solvent accessibility data 106 7 Microsecond time-resolved photo-CIDNP instrument 107 7.1 Introduction 107 7.2 Time-resolved CIDNP hardware: general considerations 109 7.2.1 Light routing 109 7.2.2 NMR sample considerations 110 7.2.3 Laser operation and synchronization 110 7.3 Hardware arrangement and operation 110 7.4 Software description 113 7.5 Examples 114 8 Relaxation processes in the high-field 19F CIDNP 119 8.1 Introduction 119 8.2 Relaxation analysis 121 8.2.1 Theoretical setup 121 8.2.2 Existing models 123 8.2.3 Proposed solution 126 8.2.4 Potential applications 132 9 References 133 10 Abbreviations 145 Chapter 1 Theoretical framework his Chapter provides the theoretical and phenomenological background for the rest of this Thesis. It is assumed that the reader is closely familiar with T the density operator description of quantum mechanical ensembles and general magnetic resonance. 1.1 Brief review of spin relaxation theory in non-viscous liquids 1.1.1. General formalism The IUPAC Gold Book [1] defines relaxation in the following way: “if a system is disturbed from its state of equilibrium, it relaxes to that state, and the process is referred to as relaxation. The branch of kinetics concerned with such processes is known as relaxation kinetics”. Relaxation and its formal description known as relaxation theory are ubiquitous in magnetic resonance. Beyond providing us with the NMR signal as such, relaxation yields valuable structural information: inter-particle distances (from Overhauser effects [2], residual dipolar couplings [3], paramagnetic shifts and relaxation rates [4]), relative orientations and angles (from cross-correlations between anisotropic interactions [5]), degrees of motion restriction as well as local and global molecular motion correlation times (from magnetic field dependence of relaxation rates [6]). Most of the spin relaxation analysis techniques rely on a remarkably compact and elegant general treatment known as Bloch-Redfield-Wangsness theory [7].