University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2014 Membrane Curvature Sorting And Generation By The Bar Domain Proteins Endophilin And Syndapin Chen Zhu University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Chemistry Commons Recommended Citation Zhu, Chen, "Membrane Curvature Sorting And Generation By The Bar Domain Proteins Endophilin And Syndapin" (2014). Publicly Accessible Penn Dissertations. 1527. https://repository.upenn.edu/edissertations/1527 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1527 For more information, please contact [email protected]. Membrane Curvature Sorting And Generation By The Bar Domain Proteins Endophilin And Syndapin Abstract Membrane curvature provides a means to control spatial organization and activity of cells. It is regulated by plenty of peripherally binding membrane proteins, including BAR domain proteins. Two important sub- families of BAR domain containing proteins are NBAR and FBAR domain proteins. However, the current understanding of BAR domain protein membrane curvature (MC) sensing and generation is insufficient. My thesis intends to contribute to alleviating this situation. We first performed curvature sorting and generation experiments of an NBAR domain protein: endophilin. We found that the endophilin NBAR domain (ENBAR) behaved as a curvature sensor or generator at different concentrations. We studied lateral diffusion of ENBAR and found its diffusion coefficients depending on its membrane density. We developed an analytical model to explain our experimental results. Our theory predicts that the membrane curvature serves as a switch that is modulated by a thermodynamic phase transition. Then we studied the influence of membrane insertion helices on NBAR domain protein MC sensing and membrane dissociation kinetics by comparing ENBAR WT protein with helices deletion mutants. We found that the two helices had essential contributions for the NBAR domain curvature sorting. The WT and mutant membrane dissociation time were positively correlated with their densities on the membrane. Irreversible binding fractions for both variants were observed. These phenomena suggest higher order structure formation of these variants on the membrane. Furthermore, we investigated the autoinhibition mechanism of full length endophilin via its H0 variants and the SH3 domain, by fluorescence experiments. We obtained evidence of the interaction between H0 helix and SH3 domain in solution and determined their binding affinities. Theseesults r experimentally support an H0/ SH3 domain mediated autoinhibition mechanism. Finally we explored the regulation of a possible autoinhibition of the tubulation ability of syndapin, an FBAR domain protein. We compared the curvature sensing, generation and initiation abilities of syndapin FBAR, full length and full length with proline rich ligand. We found that the syndapin FBAR curvature initiation ability was larger compared to the full length protein, which was likely due to the differences in their curvature sensing abilities. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor Tobias Baumgart Keywords BAR domain, biophysics, curvature, endocytosis, membrane, protein Subject Categories Chemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1527 MEMBRANE CURVATURE SORTING AND GENERATION BY THE BAR DOMAIN PROTEINS ENDOPHILIN AND SYNDAPIN Chen Zhu A DISSERTATION in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2014 Supervisor of Dissertation ________________________ Dr. Tobias Baumgart Associate Professor of Chemistry Graduate Group Chairperson ________________________ Dr. Gary A. Molander Hirschmann-Makineni Professor of Chemistry Dissertation committee Dr. Jeffery G. Saven, Associate Professor of Chemistry Dr. Paul A. Janmey, Professor of Physiology Dr. Zahra Fakhraai, Assistant Professor of Chemistry DEDICATION To Haiying Zhu and Sui Xie ii ACKNOWLEDGEMENT The completion of this thesis is not possible without the help of many others. First of all, I would like to take the opportunity to thank my thesis advisor Dr. Tobias Baugmart, who has always been very supportive since I joined the lab and served as an incredible advisor. His patience and understanding helped me adapt the research at the initial phases of my research. His enthusiasm about science always inspired me. His depth and breadth of knowledge always impressed me. His guidance helped me throughout the experimental part of my PhD studies and writing of this thesis. I am very grateful to have Dr. Jeffery G. Saven, Dr. Paul A. Janmey, Dr. Zahra Fakhraai as my committee members. I would like to thank Dr. Saven for serving as my committee chair. He has always been very kind, patient and gave me useful suggestions. Dr. Janmey shared his expertise and advice on lipid membranes with me. I am thankful that Dr. Zahra Fakhraai was willing to join my committee at the late stage of my research. I thank the past and current lab members in Baumgart’s group. I learned a lot from several senior colleagues: Dr. Aiwei Tian, Dr. Benjamin R. Capraro and Dr. Michael C. Heinrich helped me with tether pulling experiments, protein expression and purification, and the usage of optical tweezers. I also benefited from working with fellow graduate students: Tingting Wu, Zheng Shi, Zhiming Chen and other group members. It was a great pleasure and fortunate to work with so many excellent scientists. Last but not the least, I would never have been able to finish this thesis without continuous supports and love from my friends and family, especially my parents, who are always there to cheer me up and stand by me. iii ABSTRACT MEMBRANE CURVATURE SORTING AND GENERATION BY THE BAR DOMAIN PROTEINS ENDOPHILIN AND SYNDAPIN Chen Zhu Dr. Tobias Baumgart Membrane curvature provides a means to control spatial organization and activity of cells. It is regulated by plenty of peripherally binding membrane proteins, including BAR domain proteins. Two important sub-families of BAR domain containing proteins are NBAR and FBAR domain proteins. However, the current understanding of BAR domain protein membrane curvature (MC) sensing and generation is insufficient. My thesis intends to contribute to alleviating this situation. We first performed curvature sorting and generation experiments of an NBAR domain protein: endophilin. We found that the endophilin NBAR domain (ENBAR) behaved as a curvature sensor or generator at different concentrations. We studied lateral diffusion of ENBAR and found its diffusion coefficients depending on its membrane density. We developed an analytical model to explain our experimental results. Our theory predicts that the membrane curvature serves as a switch that is modulated by a thermodynamic phase transition. Then we studied the influence of membrane insertion helices on NBAR domain protein MC sensing and membrane dissociation kinetics by comparing ENBAR WT protein with helices deletion mutants. We found that the two helices had essential contributions for the NBAR domain curvature sorting. The WT and mutant membrane dissociation time were positively correlated with their densities on the membrane. Irreversible binding fractions iv for both variants were observed. These phenomena suggest higher order structure formation of these variants on the membrane. Furthermore, we investigated the autoinhibition mechanism of full length endophilin via its H0 variants and the SH3 domain, by fluorescence experiments. We obtained evidence of the interaction between H0 helix and SH3 domain in solution and determined their binding affinities. These results experimentally support an H0/ SH3 domain mediated autoinhibition mechanism. Finally we explored the regulation of a possible autoinhibition of the tubulation ability of syndapin, an FBAR domain protein. We compared the curvature sensing, generation and initiation abilities of syndapin FBAR, full length and full length with proline rich ligand. We found that the syndapin FBAR curvature initiation ability was larger compared to the full length protein, which was likely due to the differences in their curvature sensing abilities. v TABLE OF CONTENTS ACKNOWLEDGMENT ................................................................................................ III ABSTRACT ..................................................................................................................... IV TABLE OF CONTENTS ............................................................................................... VI LIST OF FIGURES AND TABLE ................................................................................. X CHAPTER 1 Introdution ................................................................................................. 1 A. Clathrin-mediated endocytosis ....................................................................................... 1 B. BAR domain containing proteins ................................................................................... 2 1. MC S&G by BAR domain proteins ................................................................................ 3 2. Endophilin ......................................................................................................................