A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Development of NMR Tools to Investigate Aggregation Phenomena Rebecca Emily Joyce Submitted for the qualification of PhD University of Sussex May 2013 University of Sussex Rebecca Emily Joyce Thesis submitted for the qualification of PhD Development of NMR Tools to Investigate Aggregation Phenomena Summary Chemistry and biology are full of examples of aggregation phenomena; from the useful applications in drug molecule stabilisation and tissue engineering, to the negative effects of causing diseases such as Alzheimer's and Parkinson's. NMR is a useful tool for probing aggregation as it can provide detail at the atomic scale; however, the molecular size of the aggregates can lead to poor resolution and spectral broadening, issues which require some development to solve. The research detailed in this thesis aims to develop NMR tools to investigate aggregation phenomena from two angles; firstly, to directly monitor aggregation using diffusion-ordered NMR spectroscopy and secondly to probe the effects of aggregates on biomimetic constructs using NMR techniques. To achieve the first aim, diffusion-ordered NMR Spectroscopy in conjunction with a size-exclusion chromatography stationary phase was developed for the purpose of monitoring aggregation in a time-resolved manner. The size-exclusion stationary phases, commonly used to separate molecules of different sizes, such as proteins from salts, in liquid chromatography methods, provide an additional perturbation of the diffusion profile of molecules of different sizes when added to NMR samples for diffusion-ordered spectroscopy analysis. The development of this method and a selection of applications are detailed within two chapters of the thesis. These studies have been published in Journal of Magnetic Resonancei and Journal of Physical Chemistry Cii. A complementary study of the interactions between aggregates and biological structures such as biomimetic cell membranes using NMR methods such as time-resolved 31P NMR and the implementation of paramagnetic shift reagents is discussed in the final two chapters. Changes in chemical shift caused by additional interactions between the shift reagent and the solvent are fully investigated and the method is applied to the study of membrane permeation by amyloid-β oligomers. iR. E. Joyce and I. J. Day, J. Magn. Reson., 2012, 220, 1{7. iiR. E. Joyce and I. J. Day, J. Phys. Chem. C., 2013, 117, 17503{17508. Acknowledgements I would first like to sincerely thank my supervisor, Dr Iain Day, for giving me the opportunity to perform the research detailed in this thesis and for the support and encouragement over the last three and a half years, as well as the multitude of corrections during the preparation of the thesis! I would also like to thank all members of the Day group, past and present, especially Matt Renshaw for providing tips on sunset yellow purification and assistance in the creation of the pulse sequence figures and Jonathan Katz for several patient explanations of how the sunset yellow absorption coefficient was determined. Much of the work involving Aβ could not have been done without the advice and expertise of my secondary supervisor, Professor Louise Serpell and members of her group, I greatly appreciate all of their assistance. In particular, Dr Tom Williams for teaching me the vesicle preparation and wash protocols, answering all sorts of questions regarding vesicle stability and acquiring some of the fluorescence data presented in the thesis. Also, Youssra Al-Hilaly for help with the preparation of Aβ and of samples for TEM imaging. On a related note, I would like to acknowledge the assistance of Dr Julian Thorpe on the acquisition of TEM images, also Dr Darren Thompson for the loan of an SEC column and Devyani Amin and Joanna Thorpe at the University of Greenwich for assistance in acquiring SEC data. Thanks must also go to all those who I have shared an office with over the three years, for providing interesting conversations, entertaining pub Fridays and amazing cakes. Especially Laura Nicholls, who ran the Marathon des Sables while I was writing this thesis and inspired me to get on with it, because writing couldn't be half as bad as running through the desert all day! In concluding these acknowledgements I need to thank my family and closest friends for all of their continuous support, despite not really understanding what my research was about. Finally, I would like to thank Gareth for the love and support throughout my PhD, particularly during the write-up process. i Contents 1 Introduction 1 1.1 Aggregation Phenomena..................................1 1.1.1 Aromatic Molecules................................1 1.1.1.1 Methods of Investigation.......................3 1.1.2 Drug Molecules..................................3 1.1.2.1 Methods of Investigation.......................4 1.1.3 Proteins......................................4 1.1.3.1 Methods of Investigation.......................6 1.1.4 Hydrogels.....................................7 1.1.4.1 Methods of Investigation.......................8 1.2 Nuclear Magnetic Resonance..............................9 1.2.1 NMR Theory...................................9 1.2.1.1 Chemical Shift.............................. 11 1.2.1.2 J-coupling............................... 12 1.2.1.3 Relaxation............................... 12 1.2.1.4 Phase Cycling............................. 14 1.2.2 Diffusion NMR.................................. 16 1.2.2.1 Effect of Magnetic Field Gradients.................. 16 1.2.2.2 Measuring Diffusion Coefficients................... 17 1.2.2.3 Pulse Sequence Adaptations..................... 19 1.2.2.4 Multicomponent Analysis....................... 20 1.2.2.5 Diffusion-Ordered Spectroscopy.................... 21 1.2.3 Paramagnetic Ions in NMR............................ 21 1.2.3.1 Paramagnetic Shift Reagents..................... 22 1.2.3.2 Paramagnetic Relaxation Agents................... 25 1.3 NMR Methods for Investigating Aggregation..................... 26 1.3.1 Changes in Chemical Shift............................ 26 1.3.2 Diffusion NMR.................................. 27 1.4 Investigating the Interactions between Aggregates and Biological Systems..... 28 1.4.1 Modelling Biological Systems.......................... 29 1.4.1.1 Phospholipid Vesicles as Biomimetic Membranes.......... 29 1.4.1.2 Physical Studies of Phospholipid Vesicles............... 31 1.4.2 Studies of Aggregate-Membrane Interactions................. 33 1.5 Thesis Outline...................................... 36 2 Materials and Methods 37 ii 2.1 Materials......................................... 37 2.1.1 Chemicals..................................... 37 2.1.2 Solvents...................................... 38 2.2 Sample Preparation................................... 38 2.2.1 Stationary Phases and Polymers........................ 38 2.2.2 Sunset Yellow................................... 39 2.2.2.1 Purification............................... 39 2.2.2.2 Sample Preparation.......................... 40 2.2.3 Phospholipid Vesicles.............................. 40 2.2.3.1 Formation By Extrusion....................... 40 2.2.3.2 Formation By Sonication....................... 40 2.2.3.3 Addition of Paramagnetic Shift Reagent............... 40 2.2.3.4 Vesicles for Calcein Leakage Experiments.............. 40 2.2.3.5 Mini-column Wash Process....................... 41 2.2.4 Fibrils........................................ 41 2.2.4.1 Insulin.................................. 41 2.2.4.2 Aβ .................................... 41 2.3 NMR............................................ 42 2.3.1 1D Experiments................................. 42 2.3.1.1 1H.................................... 42 2.3.1.2 31P................................... 42 2.3.2 Diffusion NMR Experiments.......................... 42 2.3.3 Magic Angle Spinning.............................. 43 2.3.3.1 Stationary Phase Samples for MAS................. 43 2.4 Other Physical Techniques................................ 44 2.4.1 Size-Exclusion Chromatography......................... 44 2.4.2 Dynamic Light Scattering............................ 44 2.4.3 Fluorescence Spectroscopy............................ 44 2.4.4 Transmission Electron Microscopy....................... 45 3 Diffusion NMR with Size-Exclusion Stationary Phases 46 3.1 Parameter Optimisation................................. 47 3.1.1 Concentration................................... 47 3.1.2 DOSY Parameters................................ 49 3.1.2.1 Diffusion Delay............................. 49 3.1.2.2 Gradient Duration........................... 50 3.1.2.3 Summary................................ 50 3.2 Proof of Concept Experiments.............................. 51 3.2.1 Individual Polymers................................ 51 3.2.1.1 Effect of Solvent............................. 51 3.2.1.2 Addition of Sephadex G-50...................... 52 3.2.1.3