ABSTRACT INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM by Nefertiti Muhammad The cpTat system transports fully folded proteins into the thylakoid using the proton motive force and, together, cpTatC, Tha4, and Hcf106 transport precursors across the bilayer membrane. Tha4 is predicted to form the translocation pore; however, Hcf106 may also have a role because of the large size of some cpTat substrates and its homology to Tha4. In this study, direct binding between Hcf106 and a cpTat substrate, the Oxygen Evolving Complex precursor (pOE17), was investigated. Prior work showed direct binding to Tha4 for four variants of the substrate (pOE17-D68C, -S84C, -K99C, and -T115C) using cysteine crosslinking. The current study showed little to no direct binding between Hcf106 and the last variant of the substrate (pOE17-T115C); although, that variant could interact with Tha4. Together with precursor-Tha4 interaction maps, we concluded that both cpTat components bind directly to the substrate and the strongest interactions occur at the C-tail and the C-terminal end of the amphipathic helix (APH) region. This study also investigates whether cpTat substrates stay in contact with Hcf106 throughout transport. Protease treatments were performed on crosslinked Hcf106 and pOE17 at the C-tail and N-terminal residues of Hcf106. Unexpectedly, membrane-protected and - unprotected crosslinked products were detected in the N-terminus of Hcf106. The paper presents limitations with the study and directions for future research. INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM A THESIS Submitted to the Faculty of Miami University in partial fulfillment of the requirements of the degree of Master of Science Cell, Molecular, and Structural Biology Program by Nefertiti Muhammad Miami University Oxford, Ohio 2018 Carole Dabney-Smith, Ph.D., Advisor Gary A. Lorigan, Ph.D., Reader Anne E. Hagerman, Ph.D., Reader This Thesis titled INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM by Nefertiti Muhammad has been approved for publication by The College of Arts and Science and The Cell, Molecular, and Structural Biology Program ____________________________ Carole Dabney-Smith, Ph. D. ____________________________ Gary A. Lorigan, Ph. D. ____________________________ Ann E. Hagerman, Ph. D. © Nefertiti Muhammad 2018 Table of Contents CHAPTER 1 Introduction ....................................................................................................... 1 1.1 Overview of Chloroplast Transporter Proteins .............................................................. 2 1.2 Chaperones and Regulatory Proteins .......................................................................... 4 1.3 TOC ............................................................................................................................. 5 1.31 TOC Components .................................................................................................... 5 1.32 The TOC Model ........................................................................................................ 8 1.4 TIC ............................................................................................................................... 8 1.41 TIC Components ...................................................................................................... 8 1.42 The TIC Model.........................................................................................................11 1.5 SRP ............................................................................................................................11 1.6 Secretory System........................................................................................................13 1.7 Twin Arginine Translocation System ...........................................................................14 1.8 Thesis Summary .........................................................................................................17 1.9 References .................................................................................................................17 CHAPTER 2 INVESTIGATING THE PORE COMPOSITION OF THE CHLOROPLAST TWIN ARGININE TRANSPORT SYSTEM ..........................................................................................36 2.1 Abstract ......................................................................................................................37 2.2 Introduction .................................................................................................................37 2.3 Results ........................................................................................................................39 2.31 Oxygen Evolving Complex Crosslinked Hcf106 at the C-Proximal APH and C-tail ..39 2.32 Hcf16 Crosslinked to The Precursor and Mature Protein of OE17-T115C Under Transport Conditions ..........................................................................................................42 2.4 Discussion ..................................................................................................................42 2.41 Hcf106 Integrated Interaction Map...........................................................................42 2.42 Combination of Crosslinked & Un-crosslinked OE17T115C Recovered ..................44 2.43 cpTat Transport Model: Membrane Weakening .......................................................45 2.5 Methods ......................................................................................................................47 2.51 IN VITRO SYNTHESIS OF HCF106 AND TAT SUBSTRATE CONSTRUCTS ........47 2.52 ISOLATION OF CHLOROPLASTS AND THYLAKOIDS ..........................................47 2.53 PRECURSOR RADIOLABEL-INCORPORATED TRANSLATION ...........................47 2.54 PRECURSOR INCUBATION WITH DTNB ..............................................................47 2.55 HCF106 INTEGRATION INTO THYLAKOIDS .........................................................48 2.56 TRANSPORT ASSAY .............................................................................................48 2.57 THERMOLYSIN TREATMENT ................................................................................48 iii 2.58 MEMBRANE SOLUBILIZATION ..............................................................................48 2.59 NICKEL-AFFINITY PULL-DOWN ............................................................................48 2.510 CROSSLINK-PRODUCT DETECTION ...................................................................49 2.6 References .................................................................................................................49 CHAPTER 3 Conclusions ......................................................................................................52 3.1 Conclusion ..................................................................................................................53 3.2 References .................................................................................................................55 iv List of Tables Table 2.1 INTEGRATED TABLE OF CROSSLINKING BETWEEN POE17 AND HCF106 VARIANTS ..................41 v List of Figures Figure 1.1 THE STRUCTURE AND FUNCTION OF CHLOROPLAST AND THYLAKOIDS ............................. 2 Figure 1.2 A MODEL OF TOC AND TIC. ...........................................................................................10 Figure 1.3 A MECHANISTIC MODEL OF THE CHLOROPLAST TWIN ARGININE TRANSLOCATION (CPTAT) PATHWAY. ...........................................................................................................................16 Figure 2.1 VARIANTS OF OXYGEN EVOLVING COMPLEX AND HCF106 ..................................................39 Figure 2.2 HCF106 CROSSLINKING FOR OE17-D68C AND –T115C .........................................................40 Figure 2.3 CROSSLINKING AND THERMOLYSIN ASSAY FOR OE17-T115C AND HCF106-G6C AND –I64C .....42 Figure 2.4 CHLOROPLAST TWIN ARGININE TRANSLOCATION PATHWAY MEMBRANE WEAKENING TRANSPORT MODEL .............................................................................................................46 vi Dedication For Calvin Muhammad “I loved talking with you, I miss you everyday.” -Much love from your daughter, Nefertiti vii Acknowledgments This Master’s thesis would not have been possible without the support of my graduate advisor, Dr. Carole Dabney-Smith. I am deeply thankful for her mentorship during my time in the Miami University REU program, leading me to go beyond my undergraduate education. I was more than fortunate to have her as my graduate research advisor in the Cell, Molecular, and Structural Biology program. It’s not often for graduate students to have an advisor so invested in their development as a researcher and their future after graduate school. I especially appreciate her support when I returned home for medical reasons. I could not have completed my thesis work without her patience and compassion. I feel very lucky to have worked in Dr. Dabney-Smih’s lab. In addition to her, I would like to acknowledge other faculty members, Dr. Lorigan, and Dr. Ellen J. Yezierski for their support during my time at Miami University. Tremendous amount of thanks to my lab mates, Dr. Debjani Pal, Dr. Amanda Storm, Dr. Qianqian Ma, Dr. Lei Zhang, Paul New, and Ramja Sritharan, and our lab technician, Martin Smith. I was lucky to join an amazing cohort who helped me