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Functional and Structural Analyses of a Bacterial Antifreeze Protein Chen Wang Louisiana State University and Agricultural and Mechanical College

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Functional and Structural Analyses of a Bacterial Antifreeze Protein Chen Wang Louisiana State University and Agricultural and Mechanical College Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2017 Functional And Structural Analyses Of A Bacterial Antifreeze Protein Chen Wang Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Life Sciences Commons Recommended Citation Wang, Chen, "Functional And Structural Analyses Of A Bacterial Antifreeze Protein" (2017). LSU Doctoral Dissertations. 4213. https://digitalcommons.lsu.edu/gradschool_dissertations/4213 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. FUNCTIONAL AND STRUCTURAL ANALYSES OF A BACTERIAL ANTIFREEZE PROTEIN A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Chen Wang B.S., Peking University, 2010 May 2017 i ACKNOWLEDGEMENTS I am always grateful for having Dr. Bing-Hao Luo as my major professor. He is a good mentor and provides continuous support for my PhD study and research. He is patient and gives valuable suggestions when I encounter difficulties in my experiments. He encourages and inspires me to design my own research which turns me into a better scientist. I would also like to thank my collaborators, Dr. Brent C. Christner, Dr. Svetlana Pakhomova, Erin E. Oliver, and Dr. Marcia E. Newcomer. I could not complete my thesis research without their help and efforts. I would like to express my gratitude to my thesis committee, Dr. Marcia E. Newcomer, Dr. Rui Lu, Dr. Anne Grove, Dr. Paul S. Russo, and Dr. Justin Ragains for their help and kind suggestions. It’s very kind of Dr. Ragains to join the committee as the Dean’s Representative after Dr. Russo retires. I am glad to have the chance to know and work with my previous and current lab mates. Dr. Wei Wang taught me a lot of experimental techniques when I first join the lab. Dr. Ping Hu helped me a lot with my experiments and gave thoughtful suggestions. He is also a good friend. Guannan Song and Bangqiao Yin also helped me occasionally especially when I was writing this dissertation. I want to thank Dr. Jane Reiland, Ann D. Jolissaint, Dr. William Wischusen, and Dr. Christopher S. Gregg for their help while I was teaching introductory biology labs. Last but not least, I would like to express my deep thanks to my parents Bao Guo Wang and Qiu Xiang Dong for their love and full support. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS…………………………………………………...……….….………ii ABBREVIATIONS………………………………………………………………....…………....iv ABSTRACT……………………………………..………………………………………………...v CHAPTER 1: GENERAL INTRODUCTION……………………………………………………1 CHAPTER 2: FUNCTIONAL ANALYSIS OF A BACTERIAL ANTIFREEZE PROTEIN INDICATES A COOPERATIVE EFFECT BETWEEN ITS TWO ICE-BINDING DOMAINS……………………………………………………….22 CHAPTER 3: STRUCTURAL NON-HINDRANCE OF THE ICE BINDING SITES BY A BACTERIAL MULTI-DOMAIN ANTIFREEZE PROTEIN……………43 CHAPTER 4: DIMERIZATION IS NOT REQUIRED FOR THE COOPERATIVE EFFECT BETWEEN THE TWO DOMAINS OF IBPV……………………….……….66 CHAPTER 5: GENERAL DISCUSSION……………………………………………………….75 REFERENCES…………………………………………………………………………………..78 APPENDIX: PERMISSION……………………………………………………………………..89 VITA……………………………………………………………………………………………..90 iii ABBREVIATIONS AFP: antifreeze protein AFGP: antifreeze glycoprotein BSA: bovine serum albumin CD: circular dichroism GFP: green fluorescent protein IBP: ice binding protein IBPv: ice binding protein from Vostok Station rIBPv: recombinant IBPv IBPv_ab: IBPv containing both ice binding domains but without the C-terminal region IBPv_a: the single domain protein of IBPv domain A IBPv_b: the single domain protein of IBPv domain B IBS: ice binding site PEG: polyethylene glycol RI: recrystallization inhibition TH: thermal hysteresis iv ABSTRACT Antifreeze proteins (AFPs) are a class of ice binding proteins (IBPs) that are expressed by different cold-adapted organisms to increase their freezing tolerance. AFPs have two major properties: thermal hysteresis and ice recrystallization inhibition. Here we report the functional and structural analyses of a bacterial AFP, IBPv. IBPv was originally secreted by a bacterium recovered from a deep glacial ice core drilled at Vostok Station, Antarctica. Our study showed that the recombinant protein rIBPv exhibited a thermal hysteresis of 2°C at concentrations higher than 50 µM, effectively inhibited ice recrystallization, and enhanced bacterial viability during freeze-thaw cycling. Circular dichroism scans indicated that rIBPv mainly consists of β-strands and its denaturing temperature was 53.5°C. Multiple sequence alignment of homologous IBPs predicted that IBPv contains two ice binding domains; a feature unique among known AFPs. To examine functional differences between the IBPv domains, each domain was cloned, expressed, and purified. The second domain (domain B) expressed higher ice binding activity. The 1.75 Å resolution crystal structure of IBPv was obtained, which is the first reported structure of multi-domain AFPs. The two ice binding domains are structurally similar with a RMSD of 0.68 Å by backbone alignment. Each domain consists of an irregular β-helix with a triangular cross-section and a long α-helix that parallels at one side of the β-helix. Both domains are stabilized by large numbers of tightly packed hydrophobic side chains. Structural alignment and mutagenesis studies were used to confirm a flat plane with some aligned water molecules in each domain as the ice binding site, which is located on the same face of the β-helix. Structural non-hindrance of the ice binding site was found to be pivotal for the function of IBPv. v Data from thermal hysteresis and gel filtration assays suggested that the two domains could cooperate to achieve a higher ice binding effect by forming heterodimers. Direct physical linkage of the domains was required for neither the dimerization nor the cooperative effect. However, when the dimerization of the domains was disrupted by a site mutation, the cooperative effect still remained. vi CHAPTER 1: GENERAL INTRODUCTION Ice Binding Proteins and Antifreeze Proteins Many organisms that are exposed to sub-freezing environments produce antifreeze proteins (AFPs), a sub group of ice binding proteins (IBPs), which have unique ability to inhibit ice formation by specifically interacting with ice crystals [1]. AFPs are widely found in different species including bacteria [2-4], plants [5-8], fungi [9, 10], insects [11-13], and fishes [14-16]. Generally, these proteins are classified into antifreeze proteins (AFP) and antifreeze glycoproteins (AFGP), the latter distinguished by having post-translational modifications with sugar residues [17]. Studies have shown that in extreme cold environments cells are not damaged by the low temperature but rather by ice formation inside or outside of the cells [18]. When cells are cooled slowly, ice is mainly formed in the extracellular solution. The solutes will be excluded from the ice crystals and the concentration of solutes will increase drastically [19]. Thus water will migrate out of the cells and cause the cells to dehydrate. On the other hand, at fast cooling rate cells can experience intracellular ice formation [20], which is always lethal [18]. Although the mechanism by which intracellular ice damages cells is not clear, it is proposed that the disruption of plasma membrane is the primary cause of freezing injury [21]. Secretion of AFPs allows these species to survive in cold temperatures by avoiding cell damage due to the freezing of water. Hexagonal Ice Water has many solid phases (ices), but under normal circumstances, all natural snow and ice on Earth exists in the form of hexagonal ice (ice Ih) [22], which is also the physiological 1 ligand for AFPs. The unit cell of hexagonal ice is shown in Figure 1.1A. According to Pauling’s model [23], in the hexagonal ice, each oxygen atom is tetrahedrally surrounded by 4 other oxygen atoms, and the distance between two nearest oxygen is 2.76 Å. The two hydrogen atoms in each water molecule point towards two of the four surrounding oxygen atoms to form hydrogen bonds. Even though one hydrogen atom lies between two adjacent oxygen atoms [23], this hydrogen could lie in one of the two possible positions, and therefore, as shown in Figure 1.1A, each pale sphere represents a position for hydrogen with occupancy of 0.5. This “random” distribution of hydrogens inside the ice crystals makes computer modeling of ice crystals difficult to achieve [24, 25]. Hexagonal ice contains two basal planes perpendicular to the c-axis and six equivalent side faces called prism planes (Figure 1.1B). Ice crystals may grow by adding water molecules to both basal planes and prism planes, and thus they grow freely along all directions. Figure 1.1 Illustration of hexagonal ice (Ih ice). (A) The unit cell of hexagonal ice. Oxygen atoms are labelled as red, with occupancy of 1. Hydrogen atoms are labeled as half white half gray, indicating occupancy of 0.5. (B) Illustration of hexagonal ice, with 2 basal planes and 6 prism planes. 2 Thermal hysteresis and recrystallization inhibition activities In the absence of AFPs, ice will grow rapidly in all directions when temperature
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