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Structural and Biochemical Investigations of the Mechanism Of STRUCTURAL AND BIOCHEMICAL INVESTIGATIONS OF THE MECHANISM OF HEME CAPTURE BY THE HEMOPHORE HasAp SECRETED BY PSEUDOMONAS AERUGINOSA BY GRACE JEPKORIR LAGAT B.Ed. (Sci.), Kenyatta University, 1999 Nairobi, Kenya M.Ed. (Adm.), University of Eastern Africa, Baraton, 2002 Eldoret, Kenya Submitted to the graduate degree program in Chemistry and the Graduate Faculty of the University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy Committee members: ___________________________ Mario Rivera, Ph. D. (Chair) ___________________________ Robert C. Dunn, Ph. D. ___________________________ Susan Lunte, Ph. D. ___________________________ Mikhail Barybin, Ph. D. ___________________________ Minae Mure, Ph. D. ___________________________ Wonpil Im, Ph. D. Date Defended: April 19, 2011 The Dissertation Committee for Grace Jepkorir Lagat certifies that this is the approved version of the following dissertation: STRUCTURAL AND BIOCHEMICAL INVESTIGATIONS OF THE MECHANISM OF HEME CAPTURE BY THE HEMOPHORE HasAp SECRETED BY PSEUDOMONAS AERUGINOSA Committee: ___________________________ Mario Rivera, Ph. D. (Chair) ___________________________ Robert C. Dunn, Ph. D. ___________________________ Susan Lunte, Ph. D. ___________________________ Mikhail Barybin, Ph. D. ___________________________ Minae Mure, Ph. D. ___________________________ Wonpil Im, Ph. D. Date Approved: April 19, 2011 ii ABSTRACT HasAp is a hemophore secreted by Pseudomonas aeruginosa to the extracellular media under iron limited conditions to sequester heme from the host cell. The heme HasAp complex is captured by the specific cell surface receptor HasR for subsequent internalization. X-ray and solution NMR structures of holo and apo hemophores have been solved but so far no studies have been done to elucidate the mechanism of heme loading. In the apo form, the loop bearing the distal heme iron ligand His 32 is located nearly 30 Å away from its position in the holo form. In contrast, the loop bearing the proximal ligand Tyr 75 maintains structural integrity in the apo and holo forms. This study aimed at investigating the sequential steps that lead to heme binding by HasAp and the role played by the axial-ligand bearing loops in the process of heme capture. A chromatographic method was developed to isolate and purify wild type and H32A apo HasAp. Heme binding was monitored by stopped flow spectroscopy. The results show that heme loading proceeds in two discrete kinetic phases; an initial rapid phase which entail loading of the heme onto the Tyr 75 loop followed by a slow phase where the His 32 loop undergoes a large conformational rearrangement and subsequent coordination of the heme. Molecular dynamic simulations provided more insights into the molecular motions that relay the presence of the heme in the Tyr 75 loop to the His 32 loop. To investigate the role played by the axial ligand bearing loops in heme binding, crystal structures and solution NMR studies of the apo and holo H32A-HasAp, as well as H32A- imidazole complex were carried out. The results reveal that the His 32 loop assumes a position similar to the wild type protein even in the absence of a coordinating residue. This implies that the presence of the heme in the Tyr 75 loop triggers the relocation of the His 32 loop and that this iii loop is important in protecting the macrocycle from the aqueous media. The crystal structures of Y75A and H83A show that the proteins assume the original fold although there are some conformational changes in the His 32 loop and in the secondary structure elements in regions of the protein implicated to interact with the receptor during heme release. These observations imply that information is relayed between the Tyr 75 loop and the secondary structure elements affected likely via the His 32 loop. It was also established that His 83 does not necessarily coordinate the heme in the absence of Tyr 75. Heme transfer experiments using methemalbumin as a heme source revealed that the rate of heme release from methemalbumin correlates with the rate of heme uptake by apo-HasAp. These rates are comparatively higher than the rate of heme uptake from other host heme proteins; however, they are comparable to the rate of dissociation of heme from methemalbumin in the absence of the hemophore. These results mean that methemalbumin is likely the potential target for HasAp and that heme transfers by passive diffusion and is affinity driven. iv ACKNOWLEDGMENTS First and foremost I thank God for life, wisdom, and knowledge, and for sustaining me, through it all, during the course of my studies. Northing worthwhile is ever done alone under the sun. My achievement is a worthwhile course that has taken the positive minds and caring hearts of all the people who have crossed my path ever since I took the initial step to climb the academic ladder. I am always and will ever be grateful to my dear parents for raising me and bringing me this far through their endless love, self-sacrifice, encouragement, and most of all for believing in me to make the best out of every circumstance. “Dad and Mom, I was always reassured of your great love, care, and unfailing support whenever you made a direct transatlantic call just to check on us. I reiterate my appreciation - THANK YOU SO MUCH. We made it because of you.” I am also deeply and humbly grateful to every other individual who in one way or another made it possible for me to complete this journey. I especially thank my advisor, Dr. Mario Rivera for granting me the opportunity to learn from him and work with him, and for guidance, encouragement, and continuous support throughout my studies. I am also indebted to my committee members: Dr. Robert Dunn, Dr. Susan Lunte, Dr. Mikhail Barybin, Dr. Minae Mure and Dr. Wonpil Im for all their help and advice, and for taking the time to critique my work. I owe great appreciation to the current and past members of Dr. Rivera Lab: Dr. Juan Carlos Rodriguez, Dr. Yuhong Zeng, Dr. Christopher O. Damaso, Dr. Andy Wang, Dr. Aileen Y. Alontaga, Bailey Morgan, Jordan Stobaugh, Maggie Murphy, Casey Gee, Dr. Huili Yao, Dr. Saroja Weeratunga, Yan Wang, Pavithra N. Vani, Ritesh Kumar and Kate Eshelman. Thank you v for your support, positive criticisms, and encouragement more so when I had to burn the midnight lamp working in the lab. I thank our collaborator Dr. Pierre Moënne- Loccoz for the EPR, RR, and SFS results; Dr. Scott Lovel for his helpful advice on screening for protein crystal growth and for solving the crystal structures of the proteins; Kevin. P. Battaile for collecting crystallographic data; and Dr. Wonpil Im and Huan Rui for the molecular dynamics simulation results. I also wish to thank Pr. Tony Cash and all the Lawrence (KS) and Philadelphia (IA) Adventist church members, all my relatives and neighbors, and everyone else who stood by me throughout this journey. I cannot enlist all your names but please know that your prayers, your love and your support were a constant source of strength and encouragement. To my great mentor Prof. Denford Musvosvi, and my true and exemplary friends Dr. Donita Massengill-Shaw, Dr. Jeraldine Wilson, Dr. Janet Tarus, Dr. Maru Kering, Sarah and Luka Kapkiai, Louisa Murunga, Dr. Aileen Alontaga, Rebecca Llasaca, Sayool Oh, and Dr. Jackie Obey, thank you so much for your friendship, selfless actions and kind thoughts. You were my inspiration to strive and to work harder. Last but not least I owe a lot to all my wonderful siblings: Luke, Joshua, Janet, Japheth, Damaris, Abigael, Lucy, Rupo, and Gladys. Words cannot express how much I appreciate you all for the many ways you supported me and gave me your shoulders to lean on at all times. I will never be able to give back anything in return but please know that I deeply appreciate each and every one of you. To my husband Steve, “you are one of a kind. Thank you for opening the door for me, bearing my absence from home for the longest possible period, and for enduring all the challenges to the very end.” My precious daughter Beverly, “your unfailing love, your smile, and vi your patience gave me a reason to rise up every day determined to achieve my goal. This milestone is our achievement. God has brought us this far and together we have made it!” Thank You Bev. Thank You All. vii DEDICATION To My Dear Parents (Mr. and Mrs. Eliud Lagat) viii TABLE OF CONTENTS CHAPTER I 1 INTRODUCTION….……………………………………………………………………….…...1 Iron uptake by bacteria………………………………………………….…………….…...2 Heme uptake systems in Gram negative bacteria................................................................ 4 Hemophore-dependent heme acquisition systems…...…………………..................…......6 Proposed working mechanism of hasA...............................................................................7 Structure of the model hemophore HasAs…………………………..………………….. 12 Mechanism of heme uptake in HasA……………………………..…….…………….….16 Heme binding studies…………………………………………………………………….16 RESEARCH PROBLEM AND RATIONALE…….………………………………………….....17 REFERENCES ……………………………….………………………………….…...……..…. 20 CHAPTER II 27 Structural, NMR Spectroscopic and Computational Investigation of Heme Loading in the Hemophore HasAp from Pseudomonas aeruginosa INTRODUCTION ……….……………….……………………………….…………….………27 EXPERIMENTAL PROCEDURES ….……………..……………………………......................30 Construction of H32A-HasAp ………………….…………………………….…………30 Expression and Purification of apo-proteins………………………………………….…30 Expression and Purification of Uniformly Labeled Proteins …………...………………33 Expression
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