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Single-molecule studies of protein degradation ARCHE and kinesin-8 motility MASS^C I STTUTE by by L~oEAPR 15 2015 Yongdae Shin B.S., Seoul National University (2007) LIBRARIES S.M., Massachusetts Institute of Technology (2009) Submitted to the Department of Mechanical Engineering in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Mechanical Engineering at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY February 2015 Massachusetts Institute of Technology 2015. All rights reserved. Signature redacted Author ..................... Department of Mechanical Engineering October 6, 2014 Certified by.redacted Matthew J. Lang Associate Professor of Chemical and Biomolecular Engineering, Vanderbilt University Signature redacted Thesis Supervisor C ertified by ....... ................................. Roger D. Kamm Professor of Mechanical Engineering Signature redacted Chair, Thesis Committee A ccepted by ........... ......................... David E. Hardt Professor of Mechanical Engineering Chairman, Department Committee on Graduate Studies I Single-molecule studies of protein degradation and kinesin-8 motility by Yongdae Shin Submitted to the Department of Mechanical Engineering on October 6, 2014, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Mechanical Engineering Abstract Molecular machines drive living organisms out of equilibrium and perform critical functions in almost every aspect of cellular processes. They are mechanical enzymes transducing free energy stored in chemical forms to generate motions and forces useful in cell. How these tiny machines operate in the presence of thermal agitation has been studied for a few model systems but still largely elusive. Especially, mechanisms of how molecular machines that were evolved from common ancestors diversified their machine actions to fit specific cellular requirements need to be answered. This thesis explores mechanisms of molecular machines in an effort to reveal roles of thermal fluctuations on machine functions, using two biological systems: ClpXP proteases and kinesin-8 Kif18B. As members of bigger protein families, these two fascinating nanomachines perform important cellular tasks in protein quality control and spindle formation, respectively. AAA+ ClpX unfoldases use energy from ATP binding and hydrolysis to drive mechanical unfolding and translocation of target proteins to associated peptidase ClpP. Previous ensemble biochemical and structural studies uncovered many aspects of degradation activity of ClpXP but the mechanistic understanding of ClpXP function is currently still lacking. We employed single-molecule biophysical techniques including optical trapping and single-molecule fluorescence to directly monitor unfolding and translocation activity of single ClpX hexamer as well as conformational dynamics of single subunit of ClpX. Statistical kinetic analyses on unfolding and translocation uncovered that unfolding kinetics were dominated by futile ATP hydrolysis and translocation steps contained more than one rate-limiting process for each physical stepping. Single-molecule fluorescence resonance energy transfer (sm- FRET) assay revealed dynamic switching of ClpX subunit conformations between multiple states. In the absence of nucleotide, ClpX explored available conformational spaces thermally in an erratic manner. Nucleotide binding to ClpX hexamer leads to ring contraction as well as defined hexameric conformation arrangements. Con- formational transitions were not directly coupled to ATP hydrolysis, suggesting an important role of thermal fluctuation in ClpX machine function. 3 The Kinesin-8s are plus-end directed motors that negatively regulate microtubule length. The canonical members of this kinesin sub-family showed ultra-processivity which enables Kinesin-8s to enrich preferentially at the plus-ends of microtubules to alter microtubule dynamics. Kif18B is an understudied human Kinesin-8 that also limits MT growth during mitosis. Using single-molecule assays, we found that Kif18B was only modestly processive, and that the motor switched frequently between plus- end directed and diffusive modes of motility. Measurements with truncated motors showed diffusion was promoted by a second MT-binding site located in the Kifl8B tail. Our model accounting for motility switching is consistent with autoinhibition mechanism of Kinesin-1, implying that kinesins may share common regulatory mechanisms to drive varying functional consequences. Thesis Supervisor: Matthew J. Lang Title: Associate Professor of Chemical and Biomolecular Engineering, Vanderbilt University 4 Acknowledgments Looking back on past years of graduate school, many things have happened in my life. It was not only an academic journey but also a time to look into myself. Facing the end of the graduate life, I am happy that I feel like I know what I want to do and what I should do better than I did 7 years ago. I would like to thank all people who gave me huge support and help and made my graduate life productive and enjoyable. I want to thank my advisor, Matt Lang for having me in his laboratory. When I first came to MIT, I was a naive college graduate and rather a theory-minded person. He trained and guided me to think how to precisely measure things and to build high-end instruments to accomplish that. Now I always think how better measurements can be made to tackle problems facing me. In addition to being a fantastic research advisor, Matt has been a mentor for personal stuffs as well as my career. I really appreciate his support and patience during my stay in his lab and it is just impossible to list here all his role and contribution during my graduate study. I also want to thank Bob Sauer, Tania Baker and Ryoma (Puck) Ohi for giving me a chance to work on ClpXP and Kif18B. Bob and Tania provided insightful advices on ClpXP, without them it would have been impossible to continuously make progress in my ClpX study. Puck introduced me a world of cell biology. His support and encouragement helped me to keep moving on. I am also grateful to my thesis committee, Roger Kamm and Peter So, for their commitment and critical advice during my graduate study. I was very fortunate enough to work with many talented collaborators. Joey Davis and Andreas Martin taught me how to work with ClpXP and made critical contribu- tions to make the first single-molecule fluorescence assay for ClpXP. Adrian Olivares's invaluable contribution made it possible for us to propose the mechanochemical cycles of ClpX. I also want to thank Ben Stinson, Andrew Nager and Karl Schmitz for their significant help in developing the fluorescence assay for ClpXP conformational dynamics. Sun Taek Kim introduced me to the exciting world of immunology. I am also thankful to Yaqing Du who provided most kinesins I used in my thesis and taught 5 me biology of the cell division. It has been a great pleasure to interact and share memory with the former and current Lang Lab members; Carlos Castro, David Appleyard, Ricardo Brau, Mo Khalil, Jorge Ferrer, Hyungsuk Lee, Marie Aubin-Tam, Hoi Siew Kit, Bill Hesse, Ted Feldman, Juan Carlos Cordova, Sonia Brady, Harris Manning, Yinnian Feng, Nikki Reinemann, Taishi Zhang, James Smith and Dibyendu Das. Everyday life was enjoyable due to their presence in the lab and it has been great fun to exchange scientific idea with them. I also want to thank my friends in KGSAME for their invaluable support. Finally, and most importantly, I am thankful to my family for their countless encouragement and support; many thanks to Mom, Dad and Nuna. This work is dedicated to my wife, Mihee. It was only possible with her support and love. I cannot finish without mentioning my little boy and girl; Jeongmu and Jeongwon. Their smile made me to forget all hard works in the lab and to realize what is important in my life. 6 Contents 1 Introduction 15 1.1 Molecular Machines ............................ 16 1.1.1 ClpXP Protease ........ .................. 18 1.1.2 K inesin .............................. 21 1.2 Single-Molecule Techniques .............. .. 23 1.2.1 Single-Molecule Fluorescence ...... ............. 24 1.2.2 Optical Trapping ...... ........... ........ 28 2 Monitoring conformational dynamics of single ClpX subunits 31 2.1 Sum m ary ....... ............. ............ 31 2.2 Introduction ........... ............. ........ 32 2.3 R esults .................... ............... 33 2.3.1 Single-Molecule FRET assay for ClpXP ... .......... 33 2.3.2 Nucleotide dependence of ClpX conformation ......... 35 2.3.3 Substrate dependence ...... ............ ..... 39 2.3.4 K inetics ................. ............. 39 2.4 D iscussion .......... ............ ........... 44 2.5 Materials and Methods ...... ......... ........ ... 45 2.5.1 Single-molecule fluorescence assay . ...... ..... .... 45 2.5.2 Data analysis .... ..... ..... .... ..... .... 46 3 Single-molecule kinetics of ClpXP-mediated protein unfolding and translocation 47 7 3.1 Summary . ..... .... ..... ..... .... ..... .. 47 3.2 Introduction ... ...... ..... ...... ...... .... 48 3.3 Results and Discussion .... ..... ...... ...... ... 49 3.3.1 Single-molecule optical trapping assay of ClpXP ...... 49 3.3.2 Single-molecule kinetics ................... 51 3.3.3 Multiple ATP hydrolysis cycles yet single rate limiting step . 56 3.3.4 Translocation kinetics .............. ...... 59 3.4 Materials and Methods .................. ...... 64 3.4.1 Protein constructs . .......... ........... 64 3.4.2 Single-molecule

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