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A Dissertation A Dissertation entitled Strategies for Membrane Protein Studies and Structural Characterization of a Metabolic Enzyme for Antibiotic Development by Buenafe T. Arachea Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry Dr. Ronald E. Viola, Committee Chair Dr. Max O. Funk, Committee Member Dr. Donald Ronning, Committee Member Dr. Marcia McInerney, Committee Member Dr. Patricia R. Komuniecki, Dean College of Graduate Studies The University of Toledo August 2011 Copyright © 2011, Buenafe T. Arachea This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of Strategies for Membrane Protein Studies and Structural Characterization of a Metabolic Enzyme for Antibiotic Development by Buenafe T. Arachea Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry The University of Toledo August 2011 Membrane proteins are essential in a variety of cellular functions, making them viable targets for drug development. However, progress in the structural elucidation of membrane proteins has proven to be a difficult task, thus limiting the number of published structures of membrane proteins as compared with the enormous structural information obtained from soluble proteins. The challenge in membrane protein studies lies in the production of the required sample for characterization, as well as in developing methods to effectively solubilize and maintain a functional and stable form of the target protein during the course of crystallization. To address these issues, two different approaches were explored for membrane protein studies. The first approach utilized different soluble domains as fusion partners with an alpha helical membrane protein (KcsA) to evaluate the effectiveness of this method in forming lattice contacts to produce crystals for high resolution studies. The fusion constructs were successfully cloned and expressed in C41 E. coli cells. The fusion with the maltose-binding protein (MBP) was purified and subjected to crystallization. Conditions for crystal formation and growth were identified and the MBP-fusion was iii further characterized by dynamic light scattering measurements and mass spectrometry. The second approach focuses on the extraction efficiencies of different detergent types to solubilize recombinant and constitutive membrane proteins from bacterial membranes. Using 1D gel electrophoresis for separation and MALDI-TOF spectrometry coupled with database searching for protein identification, a total of 30 unique membrane proteins including the overexpressed model protein (KcsA) were detected from the bacterial membranes. The identified proteins were isolated from the total and outer bacterial membranes, and these proteins are involved in a variety of functions which include cell respiration, ion and molecular transport, as well as in membrane biogenesis and assembly. Our study provides an initial detergent screen set that could be expanded to facilitate the selection of detergents for optimal extraction of different target membrane proteins. Aside from membrane proteins, other attractive targets for drug development include proteins that are involved in key metabolic pathways that are crucial in the survival of pathogenic microorganisms. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is located at a significant branchpoint in the aspartate biosynthetic pathway, a pathway utilized by bacterial organism to produce four essential amino acids and metabolites that serve as precursor for various cell processes. Blocking this pathway in general, and inhibiting the ASADH enzyme in particular, is fatal to microorganisms, thus raising interest in structure determination of ASADH from various families to aid in the design and development of selective inhibitors of this enzyme. The first structure of ASADH purified from a fungal species, the yeast Candida albicans, was crystallized in the presence of its nucleotide cofactor. The fungal enzyme is iv a functional dimer with similar overall fold and domain organization to its bacterial counterparts. More detailed structural comparison between the fungal and bacterial ASADHs revealed differences in secondary structural elements and in the nucleotide cofactor binding that may explain the lower catalytic activity observed for the fungal enzyme. Moreover, alterations in the dimer interface through the deletion of a helical subdomain and the replacement of amino acids involved in critical hydrogen bonding network results in the disruption of intersubunit communication channels required to support an alternating site catalytic mechanism. Elucidation of the structural details of this fungal enzyme allows an expanded assessment of ASADH as a possible target for antifungal drug development. Selective inhibitors of this fungal ASADH were identified from kinetic screening of custom made fragment libraries (Gao et al., 2010). These inhibitors were cocrystallized with the enzyme as a binary complex or a ternary complex in the presence of the NADP cofactor or the dinucleotide analog (2’5’, ADP). Co-crystallization yielded good diffraction quality crystals and these inhibitor complexes were structurally characterized by x-ray crystallography. These inhibitor studies were also expanded by evaluating ASADH targets from families of antibiotic resistant strains to help establish selectivity between different ASADH forms. v This dissertation is dedicated to my parents, my greatest source of love, strength and inspiration. Thank you for always being there. Acknowledgments I express my appreciation to my supportive adviser and mentor, Dr. Ronald E. Viola, for all the guidance during my stay in his lab. His commitment in research has motivated me to pursue my experiments despite the challenges attached with it. I appreciate his willingness to share his vast knowledge and expertise in many areas. He always makes sure that each student from his lab gets the best training not only in conducting research but also in other significant areas that would help us in our independent career as researchers. My stay in the Viola lab is one great experience that I would always value. Thank you to the past and present members of the Viola Research Group, especially to Dr. Alexander Pavlovsky and Dr. Xuying Liu. You were the two persons in the lab that I closely worked with. Thank you for sharing your time and expertise to help me in carrying out my experiments. I am also grateful to our collaborators Dr. Sami Saribas, Dr. Dragan Isailovic and Zhen Sun for all of their help in my research. I had a good time working with all of you. To my family who has supported my decision to pursue further studies abroad, I appreciate all the love and understanding. Lastly, to Jonathan Johnson, thank you for the love, encouragement and patience. You are one of my life’s sweetest blessings. vi Table of Contents Abstract……………………………………………………………………………... iii Acknowledgments…………………………………………………………………... vi Table of Contents………………………………………………………………….... vii List of Tables……………………………………………………………………….. xiii List of Figures………………………………………………………………………. xv Chapter 1 Introduction…………………………………………………………… 1 1.1 Membrane Proteins: Structure and Function ………………………………... 1 1.2 Current Methods for the Structural Determination of Membrane Proteins….. 5 1.2.1 Detergents as Tools for Membrane Protein Studies………………... 5 1.2.2 Lipidic Phase Crystallization ……………………………………… 7 1.2.3 Antibody-mediated Crystallization………………………………… 10 1.2.4 Fusion Proteins for Hydrophilic Surface Expansion……………….. 12 1.3 Aspartate Biosynthetic Pathway……………………………………………... 14 1.4 Aspartate-β-Semialdehyde Dehydrogenase………………………………….. 16 1.5 Inhibitors of ASADH………………………………………………………… 21 Chapter 2 Membrane Fusion Proteins……………………………………………. 23 2.1 Introduction…………………………………………………………………... 23 2.2 Target Membrane Fusions………………………………………………….... 25 vii 2.3 Generation of Fusion Constructs……………………………………………... 27 2.3.1 Generation of att Sites……………………………………………… 27 2.3.2 Generation of Entry Clone…………………………………………. 29 2.3.3 Generation of Fusion Protein Expression Clones………………….. 29 2.4 Pilot Protein Expression of the Fusion Constructs…………………………… 32 2.5 Cell Growth…………………………………………………………………... 37 2.6 Membrane Fusion Protein Extraction………………………………………... 37 2.7 Purification of H6-MBP-KcsA Fusion………………………………………. 38 2.8 Crystallization of H6-MBP-Fusion…………………………………………... 42 2.9 Data Collection and Structure Determination………………………………... 48 2.10 Characterization of the H6-MBP-KcsA Fusion……………………………… 53 2.10.1 DNA Sequencing of the Expression Clone………………………. 53 2.10.2 Mass Spectrometric Analysis…………………………………….. 55 2.11 Summary and Future Work…………………………………………………... 59 Chapter 3 Detergent Screening for Membrane Protein Extraction……………..... 61 3.1 Introduction…………………………………………………………………... 61 3.2 Materials and Methods……………………………………………………….. 62 3.2.1 Reagents and Chemicals…………………………………………… 62 3.2.2 Plasmid and Cells…………………………………………………... 63 3.2.3 Cell Culture and Growth…………………………………………… 63 3.2.4 Cell Membrane Preparation………………………………………... 63 3.2.5 Extraction and Solubilization………………………………………. 64 3.2.6 Gel Electrophoresis………………………………………………… 65 viii 3.2.7 MS Sample Preparation and Protein Identification………………… 65 3.2.8 MALDI-MS Analysis……………………………………………… 66 3.3 Results ……………………………………………………………………….. 67 3.3.1 Total Protein Extraction from Different Membranes……………….
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