High-resolution high-frequency dynamic nuclear polarization for biomolecular solid state NMR

Dynamic Nuclear Polarization (DNP) has exploded in popularity over the last few years, finally realizing its potential to overcome the detrimental lack of sensitivity that has plagued performing NMR experiments. Applied to magic angle spinning (MAS) experiments, this renaissance of DNP has been primarily driven by the development of instrumentation; namely gyrotron oscillators as high-power stable microwave power sources and the NMR probes and associated equipment required to spin samples routinely below 100 Kelvin. The first three chapters of this thesis provide an overview of the theory, instrumentation, and applications of DNP. Chapter 1 introduces the magnetic resonance Hamiltonian with a focus on interactions that are necessary to control in order to obtain high-resolution DNP spectra. Chapters 2 and 3 are published reviews of DNP. Whereas Chapter 2 targets magnetic resonance spectroscopists, Chapter 3 is intended for an electric engineering audience. Both reviews are included as the associated depth and coverage of the topics are complementary and lead to a better understanding of DNP. The later chapters describe in detail advancements in probe, cryogenics, and gyrotron technology required to perform DNP MAS experiments, as well as the gains in sensitivity and resolution such instrumentation has permitted. Of particular importance is the development of a cryogenic sample ejection system that resulted in exquisite resolution of spectra recorded <100 K, both of crystalline peptide and the active site of membrane proteins. Such developments in instrumentation and demonstrations of resolution go towards overturning a long-held stance in the field that DNP would always suffer from broadened, unresolved spectra. Such techniques also allow us to investigate site specific dynamics of a crystalline peptide, the high resolution SSNMR structure of which is discussed in Chapter 3. DNP and the developed instrumentation is also leveraged to measure inter-atomic distances in the active site of a membrane protein with sub-angstrom precision. Chapters 7 describes the calculation of the microwave field strength across the sample in a MAS DNP probe and introduces strategies to increase it. Finally Chapter 8 introduces a major advance in the microwave source technology associated with DNP experiments. Detailed designs of a novel 250 GHz gyrotron oscillator are shown and experiments demonstrate a continuous broad 3 GHz bandwidth with >10 W across the band, which results in substantially improved DNP performance. Thesis Supervisor: Robert G. Griffin Title: Professor, Department of Chemistry Director, Francis Bitter Magnet Laboratory DEDICATION This thesis is dedicated to my cousin, Lt. Commander Che Barnes, who perished over the Pacific Ocean in his C-130 on a search and rescue mission in 2010. ACKNOWLEDGMENTS Firstly, I want to thank Robert Griffin for creating an exciting and intense laboratory full of resources and talented, ambitious individuals to learn from and work with. Above all he fosters an environment where good ideas and technologies flourish and individuals can develop into independent scientists. Through his encouragement and support I have been able to tackle projects at graduate school I would have thought totally beyond my capabilities. He has pushed me past where I thought my limits were, and I have grown to be a better scientist and stronger person because of it. My interaction with Richard Temkin and members of his group at the Plasma and Science Center has been the singularly most exciting, challenging, and rewarding aspect of my graduate school. His caring and insightful leadership style is one I strive to emulate. His group members, Michael Shapiro, Ivan Mastovsky, Antonio Torrezan, Sudheer Jawla, Paul Woskov, Alan Cook, Elizabeth Kawalski, Brian Munroe, David Tax, and Emilio Nanni have all helped me look at DNP technology from a vacuum electronics perspective. Judith Herzfeld has been a grounding force in my struggles with instrumentation-constantly keeping my focus on a biologically relevant target. Without my exciting conversations with her discussing the ion pumping mechanism of bR I surely could not have maintained my motivation to build probes, gyrotrons, and DNP spectrometers. Jagadishwar Sirigiri is one of the most gifted teachers and innovative scientists I have had the pleasure to work with. With his guidance and instruction I was able to break through the intimidating barrier between chemistry and vacuum electronics. Without him, gyrotrons would have remained finicky, bizarrely complex black boxes. With him, I was able to improve on gyrotron design and now plan not only to use gyrotrons in my laboratory in the future, but indeed build my own and continue to develop this exciting technology. Emilio Nanni is perhaps the most promising and positive scientist I have ever met. His theoretical knowledge and experimental talents as a graduate student are unparalleled and our collaboration has been rewarding and motivating. Our interaction is what I will strive to emulate in my future collaborations. Working with Evgeny Markhasin, Loren Andreas, Eugenio Daviso, and Bj6rn Corzilius has been a pleasure and a productive time for me. Having fun running difficult experiments with them is what I always hoped graduate school would be like. Discussing science with Andy Smith and Matthew Eddy has been invaluable to my development over the years. My interaction with them demonstrates the importance of learning from and interacting with individuals who have a different skill set and knowledge base than my own. Ronald DeRocher and Ivan Mastovsky have been the most influential figures in my development as an engineer. They have tutored me in the equivalent of multiple semesters of classwork in drafting and design and emphasized the importance and utility of building high quality instrumentation right the first time through careful and meticulous planning. Patrick van de Wel, Chris Turner, Tony Bielecki, Yoh Matsuki, Melody MakJurkauskas, Jozef Lewandowski, Ga8l De Padpe, Ramesh Ramachandran, Vikram Bajaj, Marc Caporini, Marvin Bayro, Thorsten Maly, Kannan Ramaswamy, Anil Mehta, Hellmut Eckert, Marko Bertmer, and Manish Mehta have all been my instructors in developing my skills as a spectroscopist over the years and I am in their debt. Without the skills and know-how of Ajay Thakar, Jeff Bryant, Michael Mullins, and Ron DeRocher I could not have built new instrumentation and would have been stuck using commercial equipment. Blowing off steam and talking science over beers with the "Wingers" crew Leo Gomez, Becky Nicodemus, Ziad Ganim, Marc Caporini, Sean Smith, Andy Smith, Bj6rn Corzilius, Jason Locasale, Marvin Bayro, Galia Debelouchina, and Karsten Seidel has surely been a enjoyable and necessary part of my graduate school. I also thank my friends at Washington University in St. Louis. Sophia Hayes was my first instructor in magnetic resonance, is the source of my interest in sensitivity enhancement, and pointed me to MIT. I aspire to combine cutting-edge research with a positive caring mindset as she does. Jacob Schaeffer has also been a valuable resource for me to talk with and learn from over the years due to his pioneering work in MAS DNP and measuring biologically meaningful distances precisely. He is one of my professional role models. Ryan Nieuwendaal and I have discussed more science in a 72hour period than perhaps anyone can-he is an exemplary case of how much fun it can be to mix science and friendship. The chemistry faculty at Whitman College set me on my career path and showed me how fun learning and teaching chemistry can be. Allison Calhoun was my first physical chemistry instructor and emphasized the importance of having a physical intuition of the Hamiltonian. Chuck Templeton taught me attention to detail and the value in not jumping to conclusions until you process and analyze a data set completely. My family has provided me with emotional support and professional guidance over the years-without which I surely would have given up on my dreams. My sister has helped me with Matlab, and my brother is the source of my borderline obsessive attitude to writing up results in papers. My father instilled a spark and passion for science in me by showing me his experiments and writing out polymerase chain reactions on napkins over dinner when I was in grade school. Without him showing me how much science can be, I would not have a PhD from MIT or be the scientist I am today. Lastly I would like to thank my committee members, Troy van Voorhis and Andrei Tokmakoff. Their sound professional guidance and support over the last year has been extremely important to my academic career.