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Expression and Characterization of Cytochrome B5 from Giardia Lamblia

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Expression and Characterization of Cytochrome B5 from Giardia Lamblia i Expression and characterization of cytochrome b5 from Giardia lamblia A thesis submitted to the Committee of Graduate Studies in partial fulfillment of the requirements for the degree of Master of Science in the Faculty of Arts and Science Trent University Peterborough, Ontario, Canada © Copyright by Samiah Alam 2015 Environmental and Life Sciences M.Sc. Graduate Program July 15 ABSTRACT Expression and characterization of cytochrome b5 from Giardia lamblia Samiah Alam Giardia lamblia is an intestinal parasite found globally in freshwater systems that is responsible for endemic outbreaks of infectious diarrhea. As a unicellular parasite that lacks mitochondria, a respiratory chain and lives in the anaerobic environment of its host's intestine, Giardia was assumed for decades to lack heme proteins. However, its genome encodes several putative heme proteins, including three with sequence similarity to the cytochrome b5 family, referred to as Giardia cytochromes b5 (gCYT b5). Recombinant expression of one of these genes (gCYT b5-I), results in a protein (17-kDa) that is isolated with noncovalently bound heme. Resonance Raman and UV-visible spectra of gCYT b5-I in oxidized and reduced states resemble those of microsomal cytochrome b5, while sequence alignment and homology modelling supports a structure in which a pair of invariant histidine residues act as axial ligands to the heme iron. The reduction potential of gCYT b5-I measured by cyclic voltammetry is -165 mV vs the standard hydrogen electrode and is relatively low compared to those of other family members. The amino- and carboxy- terminal sequences that flank the central heme-binding core of the gCYT b5 are highly charged and do not occur in other family members. An 11-kDa core gCYT b5-I variant lacking these flanking sequences was also able to bind heme; however, we observe very poor expression of this truncated protein as compared to the full-length protein. Keywords : Giardia intestinalis, cytochrome b5, b-type cytochrome, heme/heam protein , electron transfer protein, protozoan protein, recombinant protein, molecular cloning, sequence homology, cyclic voltammetry, spectroelectrochemistry, Ultraviolet spectrophotometry, Resonance Raman, Mass Spectrometry ii ACKNOWLEDGEMENTS I would like to express sincere gratitude to my advisors Dr. Janet Yee and Dr. Steven Rafferty for allowing me to conduct research in the supportive and caring environment of their lab. Thank you for always being there to share your expertise and guide me with all aspects of graduate school. You challenged me when I needed to work hard, inspired me when I needed motivation and were patient with me when I struggled. This thesis would be impossible without your considerate and compassionate attitude. I would like to thank Dr. Ray March for teaching me mass spectrometry and sharing your knowledge and experiences. I am indebted to Dr. Shin-ichi Joseph Takayama of Dr. Grant Mauk’s lab in UBC for performing the cyclic voltammetry experiments and Dr. Logan Donaldson in York University for his assistance with NMR experiments. Special thanks to Linda Cardwell, Jane Rennie, Dr. Huda Al-Haddad, and all the graduate staff for their words of encouragement and support throughout my Trent career. Next to all members of the Yee and Rafferty lab both past and present, I thank you for creating a pleasant and friendly working environment. To Joella, Colin, Chase, Amber, Aaliya and Gail, thank you for teaching and advising. To Katie, Mirfath, Wendy, Megan, Jen, Daniel, and Elizabeth thank you for your help and enthusiasm that always created a cheerful atmosphere in the lab, it was much appreciated. To my friends Eva, Rolinda and Simanga for your friendship, emotional support, for helping me get through difficult times, and for all the happy moments we spent together. I will always treasure those times. Finally, to my husband Faraz, my siblings, and extended family thank you for your love and moral support. Most importantly, to my parents; I am truly grateful for everything you have done to raise me, support me, teach me, and give me the best life possible. iii TABLE OF CONTENTS ABSTRACT .......................................................................................................................... II ACKNOWLEDGEMENTS .................................................................................................... III LIST OF FIGURES ..............................................................................................................VII LIST OF TABLES ................................................................................................................... X LIST OF ABBREVIATIONS AND SYMBOLS ......................................................................... XI INTRODUCTION ...................................................................................................................1 1: Giardia lamblia .....................................................................................................1 1.1: Giardia lamblia life cycle .......................................................................1 1.2: Giardia lamblia metabolism .................................................................3 1.3: Giardia lamblia genome analysis ..........................................................4 2: Heme binding proteins ........................................................................................4 2.1: Flavohemoglobin ..................................................................................5 3: Cytochrome b5 .....................................................................................................7 3.1: Structural features of cytochrome b5 ...................................................7 3.2: Functions of cytochrome b5 ................................................................14 4: Recombinant Proteins .......................................................................................22 5: UV-Visible Spectroscopy of heme proteins .......................................................23 6: Electron transfer in biological systems ..............................................................25 7: Electrochemistry ................................................................................................29 7.1: Cyclic voltammetry .............................................................................34 7.2: Spectroelectrochemistry .....................................................................39 8: Electrochemical properties of cytochrome b 5 ...................................................43 9: Electrospray mass spectrometry (ESI-MS) .........................................................46 9.1: Electrospray ionization source ............................................................46 9.2: Mass analyzers ....................................................................................52 9.3: Detectors .............................................................................................53 9.4: ESI-MS spectra ....................................................................................53 10: Research objectives .........................................................................................55 MATERIALS AND METHODS ..............................................................................................56 1: Homology modelling ..........................................................................................56 2: Cloning of the full length Giardia cytochrome b 5 gene .....................................56 3: Preparation of the expression vector pET14b-gCYTb5-I ....................................59 4: Preparation of truncated cytochrome b 5 inserts ...............................................61 5: Expression and purification of recombinant gCYTb5-I .......................................62 5.1: SDS-PAGE for assessment of purity and molecular weight ................64 iv 6: Size exclusion chromatography .........................................................................65 7: Mass spectrometry ............................................................................................65 8: Spectroscopic analysis of cytochrome b 5 ..........................................................66 8.1: UV-visible spectroscopy of giardia cytochrome b5 .............................66 8.2: Resonance Raman Spectroscopy ........................................................66 9: Heme extraction of Giardia Cytochrome b 5 ......................................................67 10: Quantification of Giardia Cytochrome b 5 protein ...........................................67 11: Quantification of Heme in Giardia Cytochrome b 5 ..........................................68 12: Thermostability measurements of Giardia Cytochrome b 5 .............................68 13: pH dependence of the UV-visible spectrum of Giardia Cytochrome b 5 ..........69 14: Giardia cytochrome b 5 electrochemical measurements .................................69 14.1: Cyclic voltammetry ...........................................................................69 14.2: Spectroelectrochemistry ...................................................................70 15: NMR .................................................................................................................75 15.1: 1D 1H NMR experiments ...................................................................75 15.2: HSQC NMR experiments ...................................................................75 15.3: HSQC NMR sample preparation
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