From Slow to Ultra-fast MAS: Structural Determination of Type- Three Secretion System Bacterial Needles and Inorganic Materials by Solid-State NMR Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades "Doctor rerum naturalium" der Georg-August-Universität Göttingen - im Promotionsprogramm ProPhys der Georg-August University School of Science (GAUSS) program vorgelegt von Jean-Philippe Demers aus Saint-Jean-sur-Richelieu, QC, Canada Göttingen, 2014 From Slow to Ultra-fast MAS: Structural Determination of Type- Three Secretion System Bacterial Needles and Inorganic Materials by Solid-State NMR Dissertation zur Erlangung des mathematisch-naturwissenschaftlichen Doktorgrades "Doctor rerum naturalium" der Georg-August-Universität Göttingen - im Promotionsprogramm ProPhys der Georg-August University School of Science (GAUSS) program vorgelegt von Jean-Philippe Demers aus Saint-Jean-sur-Richelieu, QC, Canada Göttingen, 2014 Members of the Thesis defense Committee Professor Dr. Konrad Samwer (1st reviewer) I. Institute of Physics, Georg-August-Universität Göttingen Professor Dr. Christian Griesinger (2nd reviewer) Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Göttingen Dr. Adam Lange Department of NMR-based Structural Biology, Solid-State NMR Spectroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen Professor Dr. Helmut Grubmüller Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen Professor Dr. Hans Hofsäss II. Institute of Physics, Georg-August-Universität Göttingen Professor Dr. Markus Zweckstetter German Center for Neurodegenerative Diseases (DZNE), Göttingen Date of the oral examination: April 23rd 2014 ii iii Affidavit I hereby declare that this thesis has been written independently and with no other sources and aids than quoted. Furthermore I confirm that this thesis has not been submitted as part of another examination process neither in identical nor in similar form. ____________________ __________________ Jean-Philippe Demers Place, Date iv "I have not yet lost a feeling of wonder, and of delight, that this delicate motion should reside in all the ordinary things around us, revealing itself only to him who looks for it. I remember, in the winter of our first experiments, just seven years ago, looking on snow with new eyes. There the snow lay around my doorstep - great heaps of protons quietly precessing in the earth’s magnetic field. To see the world for a moment as something rich and strange is the private reward of many a discovery." — Edward M. Purcell, Research in nuclear magnetism, Nobel lecture, Dec. 11th 1952 À mes parents, v Acknowledgements I would like to specially thank my supervisor Adam Lange for his support throughout my Ph.D., by being a constant source of motivation and new interesting ideas and by being a nice supervisor with many human qualities: tolerance, openness and patience. I had the opportunity to work in an environment of exceptional scientific richness at the Max-Planck Institute for Biophysical Chemistry in the NMR-based Structural Biology department and I want to thank Christian Griesinger for creating and organizing this environment. I am extremely grateful to the members of my research group in Solid-State NMR: Vinesh Vijayan, Robert Schneider, Karsten Seidel and Antoine Loquet had the patience and the generosity to teach me the practical aspects of solid-state NMR directly at the instrument. I also want to thank all my colleagues of the group over the years for their friendship and their conversations: Ashutosh, Zrinka, Guohua, Veniamin, Pascal, Sheng-Qi, Venita, Hannes, Nils, Chaowei, Songhwan, Birgit, Benni, Suresh, Ann- Christin. I am very grateful to Ann-Christin Poeppler for the proofreading of my thesis. I have special thanks for Venita for helping me improve my spoken German, and for Antoine and Hannes for many interesting discussions on solid-state NMR. I want to thank Vinesh, Zrinka, Gitta, Birgit and Hannes for their help in the maintenance of the 800 MHz ssNMR spectrometer. Many interesting projects were possible due to the help of our collaborators. I want to thank Rajendra S. Ghadwal, Shaban Khan, Sakya S. Sen, Amit Pratap Singh, Prinson Samuel and Herbert W. Roesky of the Institute of Inorganic Chemistry at the Georg-August-Universität Göttingen for providing us many interesting inorganic vi compounds. I want to thank Jacek Biernat and Eckhard Mandelkow at the Max-Planck Unit for Structural Molecular Biology at DESY in Hamburg for providing us tau samples. I want to thank Yusuke Nishiyama and Michal Malon at JEOL RESONANCE Inc in Tokyo for carrying experiments at ultra-fast MAS. For my research project on the study of the T3SS needle, I want to thank Birgit Habenstein for her great help on the project, Antoine Loquet and Suresh Kumar Vasa for technical assistance and discussion on this topic, Christian Griesinger and Arturo Zychlinsky for the initial support of the project, and Brigitta Angerstein for expert technical assistance. I want to thank Nikolas G. Sgourakis at the Laboratory of Chemical Physics, NIDDK/NIH, Bethesda MD and David Baker at the University of Washington in Seattle, WA for a very active collaboration concerning the modelling of structural data, Rashmi Gupta, Michael Kolbe and Britta Laube at the Max Planck Institute for Infection Biology in Berlin for carrying in vivo experiments on bacteria, Dietmar Riedel at the MPIbpc in Göttingen for recording electron microscopic images of polymerized needles, Gregory Bubnis and Helmut Grubmüller for discussions on computational simulations of T3SS needle functions. Foremost, I want to thank and highlight the work of Karin Giller and Stefan Becker in our department at the MPIbpc in producing biological samples of unsurpassed quality for our solid-state NMR studies. I also want to thank Stefan Becker for keen and crucial discussions of our structural studies. I want to thank the Defense committee and the support of Konrad Samwer over the course of my Ph.D. I want to acknowledge the financial support of the Natural Sciences and Engineering Research Council of Canada (NSERC/CRSNG) of Canada and the Max Planck Society. vii Finally, on the personal side, I want to thank the support and friendship of the members of the NMR-based Structural Biology Department: Davood, David, Hari, Adam, Sibelle, Rakhi, Pablo, Luigi, Hessam, Marta, Han, Guohua and many others. My interest in science has always been highly supported and nurtured in my school, college and university time. For this I want to thank my science teachers and mentors Martin Bisaillon, Gilles Dumas and Linda Brunet at École secondaire Marcellin- Champagnat, Céline Dupont at Collège Édouard-Montpetit, Éric Rassart at UQÀM, Hugh Clark, Moshe Szyf and Anthony Mittermaier at McGill University. I had the chance to develop my interest for science through science fair competitions. I want to thank those who provide teenagers this wonderful opportunity: I especially want to thank the work of Marie-Hélène Deneault, Jean Verville, Carole Charlebois and Marthe Poirier at the Conseil de développement du loisir scientifique (CDLS) and Conseil du Loisir Scientifique de la région Métropolitaine (CLSM) who had a positive impact on my interest in a scientific research life. Je souhaite remercier mes parents Diane et Roland Pierre, ma soeur Karoline et son copain Xavier, ainsi que mes amis Jasmin, Veronik et Willy pour leur essentiel soutien moral. My friends Tatiane, Peipei and Chuli have been encouraging me and for this I am grateful. I want to thank the support, listening and companionship of Xueqin Zeng. viii Contents Part I: Introduction to Solid-State NMR ............................................................................. 1 1. Introduction to Solid-State NMR ................................................................................. 1 1.1. Nuclear spin ................................................................................................................... 1 1.2. Interaction with external magnetic fields .................................................................. 3 1.2.1. Spin-lock RF pulse ...................................................................................................... 5 1.3. Chemical shielding ........................................................................................................ 6 1.4. Prediction of Chemical Shift Anisotropy (CSA) tensor values ............................... 8 1.5. Dipolar couplings .......................................................................................................... 9 1.5.1. Measurement of inter-nuclear distances ............................................................... 10 1.5.2. Measurement of molecular motions ...................................................................... 11 1.6. Magic-Angle Spinning ................................................................................................ 11 1.7. Isotopic labeling ........................................................................................................... 14 1.8. References ..................................................................................................................... 16 Part II: Ultrafast Magic-Angle Spinning and Low-Power Sequences ......................... 19 2. Introduction to ultra-fast MAS spinning solid-state NMR ................................... 19 2.1. Summary .....................................................................................................................