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Ahmad Haniff Jaafar Modification of the Prosegment in Understanding its Role in the Folding and Function of PMII by Ahmad Haniff Jaafar A Thesis Presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science Guelph, Ontario, Canada © Ahmad Haniff Jaafar, August, 2014 ABSTRACT MODIFICATION OF THE PROSEGMENT IN UNDERSTANDING ITS ROLE IN THE FOLDING AND FUNCTION OF PMII Ahmad Haniff Jaafar Advisor: University of Guelph, 2014 Professor R.Y. Yada This thesis explores the folding and activation of plasmepsin II (PMII) and the role of its prosegment (PS) on structure – function. Three different constructs of PMII were designed with PSs of varying lengths: extended PSPMII with a 60 residue PS, truncated PSPMII with a 48 residue PS, and NoProPMII with no PS. Extended and truncated PSPMII produced mature enzyme with similar conformation. NoProPMII mature, however, showed improper folding as indicated by low thermal stability, a more solvent-exposed conformation, an 11-fold reduction in the activity assay, and a lower pepstatin-A requirement for complete inhibition. In addition, the PS length was discovered to affect the activation of PMII. Extended PSPMII produced mature enzyme with an extra two PS residues (+2 PMII mature) whereas truncated PSPMII produced mature enzyme with an extra 12 PS residues (+12 PMII mature). The role of a PS in PMII folding was further investigated by conducting folding kinetic studies on PMII. It was found that the native PMII (Np) did not fold at the lowest free energy, but was kinetically stabilized. Upon unfolding, Np formed a thermodynamically stable, yet inactive refolded state (Rp). Np was characterized to have a slow rate of unfolding and folding, as indicated by large free energy barriers to unfold and fold of 24.50 and 25.12 kcal/mol, respectively. In the presence of the extended PS (60 residues), the energy landscape was shifted and the activation energy barrier was lowered to 12.37 kcal/mol, which enhanced the folding rate by approximately 18,550 times. To elucidate the effect of PS residue length on PMII folding, structural analysis and in silico simulation were conducted on the two zymogens of PMII: extended PSPMII and truncated PSPMII. Both zymogens appeared to be in a more solvent-exposed conformation as compared to their mature forms. Due to the oppositely charged residues between the main protein body and the PS, it appeared that the interactions between both structures were driven by electrostatic forces with 27 and 34 interactions (H-bonds and salt-bridges) measured between the main PMII body and truncated and extended PS, respectively. ACKNOWLEDGEMENTS It gives me great honour to acknowledge herein Prof. Rickey Yada for his intellectual guidance and unceasing support rendered towards me during my Ph.D. candidature at University of Guelph. My time at Guelph has been a truly remarkable journey of self exploration and knowledge enrichment best indicated by the completion of my studies, and presentation of this thesis thereafter. Million thanks also go to my Advisory Committees Prof.Yoshinori Mine, Prof. George Harauz and Prof. Alejandro Marangoni who have equally contributed in making my success achievable. My deepest and most sincere gratitude is thus conveyed to my family and parents. Their love and compassion have always ignited the passion in me to strive and persevere in order to make it to the finishing line. As such, my success is as much as it is theirs. Great thanks to my colleagues, especially all my lab mates Huogen, Brian, Yasumi, Dref, Derek, Charity, Brenna and Reena for their help during my time in Guelph. My appreciation is also extended to Mary Anne Smith for her expert editing throughout my entire thesis. And last but never least, to my employer, Universiti Putra Malaysia (UPM), and my sponsor, Malaysian Ministry of Education (MOE), I am most grateful for and thus fully acknowledge the chance and opportunity given to me. My Ph.D. would not be completed without the financial support and endorsement from both parties. THANK YOU. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................................ ii ACKNOWLEDGEMENTS ................................................................................................................... iv TABLE OF CONTENTS ........................................................................................................................ v LIST OF TABLES ............................................................................................................................... viii LIST OF FIGURES ............................................................................................................................... ix LIST OF ABBREVIATIONS ................................................................................................................ xi CHAPTER 1 Introduction and literature review ................................................................. 1 1.1 Introduction ................................................................................................................................ 1 1.2 Literature Review ....................................................................................................................... 5 1.2.1 The family of APs ................................................................................................................. 5 1.2.2 General structure and mechanism of APs ............................................................................. 7 1.2.3 Multifunctional PS in APs ................................................................................................... 10 1.2.4 Prosegment catalyst folding ................................................................................................ 12 1.2.5 Prosegment catalyzed folding in APs .................................................................................. 13 1.2.6 Malarial APs ........................................................................................................................ 14 1.2.7 Plasmepsin II ....................................................................................................................... 15 1.2.8 Expression of PMII ............................................................................................................. 16 1.2.9 Unique characteristic of the PMII PS .................................................................................. 17 CHAPTER 2 Characterization of PMII with various lengths of prosegment ................. 19 2.1 Introduction .............................................................................................................................. 19 2.2 Materials and Methods ............................................................................................................ 20 2.2.1 Materials .............................................................................................................................. 20 2.2.2 Generation of PMII expression constructs .......................................................................... 20 2.2.3 Expression and purification of PMII constructs .................................................................. 22 2.2.4 Purification of PMII constructs ........................................................................................... 22 2.2.4.1 Extended PSPMII and truncated PSPMII ................................................................. 23 2.2.4.2 NoProPMII ................................................................................................................ 24 2.2.5 Protein concentration determination of PMII constructs and mature enzymes ................... 24 2.2.6 Optimum pH of activity and stability of PMII mature enzymes ......................................... 25 2.2.7 Structural characteristic analysis of PMII mature enzymes ................................................ 26 2.2.7.1 Far-UV circular dichroism spectroscopy.................................................................... 26 2.2.7.2 Intrinsic fluorescence spectroscopy ............................................................................ 26 2.2.8 Thermostability analysis using differential scanning calorimetry ....................................... 26 2.2.9 Kinetic parameters of PMII mature enzymes ...................................................................... 27 2.2.10 Active enzyme comparison of PMII mature enzymes ...................................................... 27 2.2.11 Structural prediction of +2 PMII and +12 PMII mature enzymes ..................................... 28 2.2.12 Statistical Analysis ............................................................................................................ 28 2.3 Results and Discussion ............................................................................................................. 28 2.3.1 Construction and soluble expression of PMII constructs .................................................... 28 2.3.2 Purification of PMII constructs ........................................................................................... 35 2.3.3 Activation to +2 PMII, +12 PMII, and NoProPMII mature enzymes ................................. 43 2.3.4 Optimum pH of activity and stability of PMII mature enzymes ......................................... 45 2.3.5 Structural characteristics of PMII mature enzymes............................................................
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