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Functional Analyses of Human Serum Paraoxonase1 (Hupon1) Mutants Using

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Functional Analyses of Human Serum Paraoxonase1 (Hupon1) Mutants Using Functional Analyses of Human Serum Paraoxonase1 (HuPON1) Mutants Using Drop Coating Deposition Raman Difference Spectroscopy THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Hua Ying Graduate Program in Chemistry The Ohio State University 2010 Master's Examination Committee: Professor Terry L. Gustafson, Advisor Professor Thomas J. Magliery Copyright by Hua Ying 2010 Abstract We present work on the structural implications of specific mutants of Paraoxonase1 (PON1) G2E6, and the turnover rate upon bonding of the enzymes with paraoxon when compared to the wild-type enzyme by using vibrational spectroscopy. A new Raman spectroscopy called Drop Coating Deposition Raman (DCDR) is utilized in our work. The Raman band changes in the paraoxon/H115W system are in good agreement with computational calculations and are strong evidence of the formation of the paraoxon hydrolysis product, p-nitrophenol in the reaction system. The corresponsive turnover rates of G2E6 wild-type and its two mutants, H115W and H115T, are also observed in DCDR spectra. ii Dedication This document is dedicated to my friends and family. iii Acknowledgments I would like to thank my advisor, Prof. Terry Gustafson for his encouragement, support, guidance, and motivation. I also wish to thank our collaborators Prof. Thomas Magliery, Prof. Christopher Hadad and the members of their groups for assistance on the U54 project. I would like to thank Rachel Baldauff for going through all the U54 program meetings with me. I also want to thank Lynetta Mier for helping me with editing my thesis in every detail. I wish to thank Nicole Dickson and all my labmates for being such a great family for me during these three years. Special thanks go to Judy Brown for all those talks and understanding when I was feeling low. I want to thank Jinquan Chen for being such a wonderful friend in these years. I would not be so clear with my life goal without their understanding, support, and encouragement. I also would like to thank Yibo Zhang, Xiaoyan Guan, Jing Li, Yehong Shen, Jie Wang, Simon Pondaven, Yawen Zhen, Xiaojing Li, and many of my friends all over the world for making these three years in U.S. to be such a wonderful experience. Finally, I would like to thank my parents for understanding and supporting all my decisions, and giving me all their love. Also, very special thanks go to my husband Ruihua Bian. Thanks for understanding and supporting all my choices and always being there for me. iv Vita June 14, 1984……………………Born – Shanghai, China July 2007………………………..B. S. Chemistry, Fudan University, Shanghai, China September 2007 – May 2008……Graduate Assistant, The Ohio State University June 2008 – present…………......Graduate Teaching Assistant, The Ohio State Universiy Fields of Study Major Field: Chemistry v Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Figures .................................................................................................................. viii List of Tables ...................................................................................................................... xi CHAPTER 1 INTRODUCTION ..................................................................................... 1 1.1 Human Serum Paraoxonase1 (HuPON1)............................................................ 1 1.2 Paraoxon ............................................................................................................. 4 1.3 Raman Spectroscopy in Enzymology ................................................................. 6 1.4 Raman Difference Spectroscopy ......................................................................... 8 1.5 Drop Coating Deposition Raman (DCDR) Spectroscopy .................................. 9 CHAPTER 2 EXPERIMENTAL ................................................................................... 12 2.1 Samples ............................................................................................................. 12 2.2 Instrument setup ................................................................................................ 12 2.3 Procedure .......................................................................................................... 14 2.4 Spectra Smoothing ............................................................................................ 17 2.5 Spectra Subtraction ........................................................................................... 17 vi CHAPTER 3 RESULTS AND DISCUSSION .............................................................. 18 3.1 Comparison of Solution, Single Crystal and DCD Raman Spectroscopy ........ 18 3.2 Reproducibility of DCDR Method.................................................................... 20 3.3 Paraoxon/PON1 System Studies ....................................................................... 22 CHAPTER 4 CONCLUSIONS ..................................................................................... 35 4.1 Paraoxon/PON1 System Studies ....................................................................... 35 4.2 Future Directions .............................................................................................. 35 vii List of Figures Figure 1.1 .......................................................................................................................... 2 Overall structure of PON1. a) View of the six-bladed b-propeller from above. Two calcium atoms in the central tunnel of the propeller (Ca1, green; Ca2, red). b) A side view of the propeller, including the three helices at the top of the propeller (H1-H3). Figure 1.2 .......................................................................................................................... 3 a) A schematic representation of the overall architecture of the active site of HuPON1. The ester/lactone binding region (in cyan) and paraoxon binding region (in yellow) are shown as circles, and the calcium-ligating center with postulated catalytic residues His dyad and D369 are shown in the red region. b) Superposition of three substrates phenylacetate (cyan), d-valerolactone (green), and paraoxon (yellow) bound in the active site of HuPON1. The proposed catalytic residues, His dyad are shown in blue. Figure 1.3 . .......................................................................................................................... 4 Structure of G2E6 PON1. Residue H115 is proximal to the “catalytic” Ca2+ and phosphate ion found in the crystal. Figure 1.4 . .......................................................................................................................... 5 The hydrolysis reaction of paraoxon. Figure 1.5 .......................................................................................................................... 6 The binding conformation of paraoxon with HuPON1 wild-type. Figure 1.6 ........................................................................................................................ 10 A 10x microscopic image of the DCD of G2E6 H115K. Figure 2.1 . ........................................................................................................................ 13 Microscope Raman setup. Figure 2.2 ........................................................................................................................ 15 A 50x Microscope image of DCD paraoxn/G2E6 H115W system at ratio 1:500 Figure 2.3 ........................................................................................................................ 15 a) A 5x microscope image of DCD 5 mM lysozyme. The crystal of salt in buffer is observed. b) The DCD Raman spectrum of Tris-HCl buffer. Asterisks indicate the major bands of buffer. Figure 2.4 ........................................................................................................................ 16 A 20x microscope image of the single crystal of lysozyme. viii Figure 3.1 ........................................................................................................................ 18 Comparison of different Raman spectra of lysozyme. a) The spectrum of 50 mM saturated lysozyme in 50mM Tris-HCl buffer pH=8. b) The spectrum of lysozyme single crystal. c) The spectrum of DCD lysozyme in 50mM Tris-HCl containing 50 mM NaCl and 1 mM CaCl2 at pH 8. Figure 3.2 ........................................................................................................................ 21 Reproducibility of DCD protein Raman spectra. a) and b) Average spectrum of 5 acquisitions of two different G2E6 H115W depositions. c) Difference spectrum between trace a and b. d) Spectrum of buffer 50mM Tris-HCl containing 50 mM NaCl and 1 mM CaCl2 at pH 8. Figure 3.3 ........................................................................................................................ 22 Reproducibility of DCD small molecules Raman spectra. a) and b) Average spectrum of 5 acquisitions
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