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A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio Molitor

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A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio Molitor A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio molitor A thesis presented to the faculty of the College of Arts and Sciences of Ohio University In partial fulfillment of the requirements for the degree Master of Science Young Eun Choi November 2007 2 This thesis titled A Study on the Hyperactive Antifreeze Proteins from the Insect Tenebrio molitor by YOUNG EUN CHOI has been approved for the Department of Physics and Astronomy and the College of Arts and Sciences by Ido Braslavsky Assistant Professor of Physics and Astronomy Benjamin M. Ogles Dean, College of Arts and Sciences 3 Abstract CHOI, YOUNG EUN, M.S., November 2007, Physics and Astronomy Title: A study on the hyperactive antifreeze protein from the insect Tenebrio molitor (51 pp.) Director of Thesis: Ido Braslavsky Antifreeze proteins (AFPs) are class of proteins that protect organisms from damages caused by freezing, either by preventing freezing or minimizing frost damages. AFPs effectively lower the temperature at which water freezes. They are classified by the depression of the freezing temperature compared to the melting temperature, i.e. Thermal Hysteresis activity (TH): moderately active AFPs and hyperactive AFPs. It is still unknown what makes some AFPs hyperactive compared to the much less active classes of AFPs. Previous studies showed that fusion proteins of fish type III AFP bind independently to ice. This conclusion was derived from experiments with bulky proteins that were fused to this moderately AFP. One possible explanation for the increased activity of the hyperactive AFPs is that they might function cooperatively. To investigate this, the hyperactive AFP from the mealworm, Tenebrio molitor (TmAFP), was linked to bulky proteins. In this thesis, these fusion proteins were assayed by a nanoliter osmometer, a device that has been designed to measure TH of AFPs. The results indicate that the addition of large molecules to the TmAFP does not induce any loss of thermal hysteresis activity; these fusion proteins were rather more active than free TmAFP at almost all concentrations. Further, ice crystal morphologies obtained by the fusion proteins were the same as the ones of free TmAFP. Therefore, it is concluded that TmAFPs independently bind to ice and their enhanced thermal hysteresis activity does not result from cooperativity. Approved: _____________________________________________________________ Ido Braslavsky Assistant Professor of Physics and Astronomy 4 Acknowledgements I would like to acknowledge and thank my advisor, Dr. Ido Braslavsky, for the excellent guidance and the considerable help with numerous discussions. His support with patience and generosity encouraged me. For our collaboration, I am grateful to Dr. Peter Davies from Queen's university and to Dr. Deborah Fass and Maya Bar from the Weizmann institute of science, for providing proteins that I used for my experiments and for a lot of useful comments about the proteins. I would also like to thank Dr. Natalya Pertaya and Yeliz Celik for their assistant and suggestion while I was measuring the activity of AFPs. Finally, I thank my mother for her support and endless love. Without the support of my family, I could not finish this work. Thanks to all my friends for encouragement throughout my studies. Table of Contents Abstract............................................................................................................................... 3 Acknowledgments............................................................................................................... 4 List of Figures..................................................................................................................... 7 1. Introduction and literature review................................................................................... 8 1.1 What are antifreeze proteins?..................................................................................... 9 1.2 Ice structure.............................................................................................................. 10 1.3 The general mechanisms of antifreeze activity........................................................ 11 1.3.1 Colligative phenomena ....................................................................................... 12 1.3.2 Adsorption-inhibition: Gibbs-Thomson (Kelvin) Model.................................... 12 1.3.3 Nucleation inhibition .......................................................................................... 17 1.4 Molecular structures of antifreeze proteins.............................................................. 18 1.5 Hyperactive antifreeze proteins ............................................................................... 22 1.6 Previous studies of type III AFP with attached bulky protein................................. 23 1.7 Aim of the thesis ...................................................................................................... 26 2. Sample preparation and experimental methods ............................................................ 27 2.1 Materials .................................................................................................................. 27 2.1.1 Tenebrio Molitor AFP (TmAFP)........................................................................ 27 2.1.2 MBP-His6-TEV-TmAFP.................................................................................... 27 2.1.3 His6-eGFP-TmAFP ............................................................................................ 29 2.2 Experimental Equipment ......................................................................................... 30 2.3 Experimental procedures and measurements........................................................... 32 2.3.1 Protein purification by adsorption to ice............................................................. 32 6 2.3.2 Procedure and measurement of protein concentration........................................ 35 2.3.3 Procedure and Measurement of the Thermal Hysteresis activity ....................... 36 3. Results........................................................................................................................... 38 3.1 Ice crystal growth and morphology as a function of the TmAFPconcentration...... 38 3.2 Thermal hysteresis activity of TmAFP-fusion proteins........................................... 40 4. Conclusion .................................................................................................................... 43 5. Discussion and future work .......................................................................................... 45 References......................................................................................................................... 47 7 List of Figures Figure 1: Ice structure .....................................................................................................11 Figure 2: Illustration of the thermal hysteresis...............................................................16 Figure 3: Adsorption-Inhibition of ice crystal growth....................................................16 Figure 4: A schematic representation of the curvature of the ice developed between adjacent AFP molecules..................................................................................................17 Figure 5: The structures of AFPs....................................................................................20 Figure 6: The structure of TmAFP..................................................................................21 Figure 7: Ice crystal morphologies of insect AFP and fish AFP ....................................23 Figure 8: Thermal Hysteresis of fish type III AFP and its fusion proteins.....................25 Figure 9: Ice crystals formed in the presence and absence of AFP-fusion proteins and their constituents .............................................................................................................26 Figure 10: A schematic representation of MBP-His6-TEV-TmTHP (TmAFP).............29 Figure 11: A schematic representation of His6-eGFP-TmTHP (TmAFP) .....................29 Figure 12: The experimental apparatus...........................................................................31 Figure 13: Screen shot of the Labview interface for controlling the temperature of the ice crystals.......................................................................................................................32 Figure 14: The cold finger apparatus..............................................................................34 Figure 15: Thermal hysteresis activity (°C) vs. concentration (μM) for TmAFP ..........39 Figure 16: Growing direction of fish AFP and insect AFP ............................................39 Figure 17: Ice crystal morphologies ...............................................................................41 Figure 18: The thermal hysteresis activity of TmAFP and TmAFP-fusions..................42 Figure 19: Schematic representation of cooperativity and non-cooperativity ...............44 8 1. Introduction and literature review A number of different types of proteins have evolved in fish [1], insects [2], plants [3,4], and other organisms [5-7] to interfere with ice crystal growth. The proteins that protect the organisms by preventing or reducing the damage caused by freezing are called “antifreeze proteins” (AFPs), and also known as “thermal hysteresis proteins” (THPs), “ice
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