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The Investigation of Peptide and Protein-Glycosaminoglycan Binding The Investigation of Peptide and Protein-Glycosaminoglycan Binding Interactions using Fluorescent Probes By Anthony F. Rullo A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Chemistry University of Toronto Copyright by Anthony F. Rullo 2012 Abstract The Investigation of Peptide and Protein-Glycosaminoglycan Binding Interactions using Fluorescent Probes Doctor of Philosophy Graduate Department of Chemistry University of Toronto Anthony F. Rullo 2012 The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide- heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated ii in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide- based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples. iii Acknowledgments I would sincerely like to thank Professor Mark Nitz for the incredible opportunity to pursue my doctoral studies under his supervision. Your mentorship and scientific ability has had a very positive and profound impact on my development as a graduate student and on my transition from a student to a young scientist. I am also very thankful to the members of my committee professor Rebecca Jockusch and professor Andrew Woolley for their invaluable advice and support. Thank you to the members of the Nitz group and especially to Rodolfo Gomez. I have very much enjoyed our numerous insightful discussions ranging from kinetics and organic synthesis to the everyday trials and tribulations of life as a doctoral student. I wish you the best of luck in your future endeavors and I know that you will always be ready to replace luck with impressive perseverance and technical skill should the situation require. A special thanks to my friend Andrew Beharry who’s ability as a doctoral student and passion for science has been an inspiration throughout my studies at the University of Toronto. It has been a pleasure developing as a doctoral student in chemistry alongside you and I very much look forward to being a colleague of yours in the future. The work presented in this thesis is the culmination of five years of dedication, dogged determination, and devotion and is a testament to the special people in my life who introduced me to these virtues. My dad and first mentor John, who personifies strength and discipline, taught me at an early age the importance of staying the course and the meaning of endurance. I learned by his example to never quit and to persevere in the face of adversity. Anything I ever have achieved or will achieve in life is attributed to him and his example. My mother Anna’s selflessness and sacrifice throughout the years made this PhD possible. I will be forever indebted to you and cannot describe with words the extent of my gratitude and appreciation. To my Amy, your love and support has been an integral part of my doctoral studies. You have been on the front line alongside me throughout this entire process from the frequent frustrations, to the joy of making exciting discoveries and positive contributions to the field. I can’t imagine having done this without you. iv I would also like to give a special acknowledgment to my nanna Serafina. I could never do justice in describing the magnitude of the significance you have had in my life and the quality of the human being you are by simply putting black letters down on a white page. Through the many adventures of growing up, I could never have hoped for a more nurturing, loyal, and noble protector and friend than the one God gave me as a grandmother through you. Your devotion to your grandchildren was not in vain and is largely responsible for the many successes we have all enjoyed including the attainment of my doctoral degree in chemistry and the work presented in this thesis. It is with true sadness I bring this chapter of my life to a close without being able to share it with you and without you deriving the pleasure from it that you, most of all, deserved to enjoy. You will serve as an inspiration to me always and will forever be a critical part of the foundation upon which I am built. This thesis is dedicated to you. v Table of Contents Abstract……………………………………………………………………………………. ii Acknowledgments…………………………………………………………………………. iv Table of Contents…………………………………………………………………………. vi List of Abbreviations……………………………………………………………………… viii List of Tables………………………………………………………………………………. x List of Figures……………………………………………………………………………... xi List of Schemes……………………………………………………………………………. xiv Chapter 1. Introduction………………………………………………………………….. 1 1.1 Carbohydrates in glycobiology…………………………………………………………. 1 1.2 Glycosaminoglycans……………………………………………………………………. 3 1.3 Heparin and heparan sulfate……………………………………………………………. 5 1.4 Lectins………………………………………………………………………………….. 6 1.5 Lectin-carbohydrate interactions……………………………………………………….. 8 1.6 Heparin/HS-Protein Interactions……………………………………………………….. 10 1.7 Techniques to study HS/heparin interactions…………………………………………... 15 1.8 The development of probes for HS/heparin……………………………………………. 16 1.9 Purpose of study……………………………………………………………………….. 22 References………………………………………………………………………………….. 23 Chapter 2. The effects of the spatial display of positive charge on peptide heparin binding affinity…………………………………………………………………... 27 2.1 Introduction……………………………………………………………………………. 27 2.2 Results………………………………………………………………………………….. 30 2.3 Discussions and Conclusions…………………………………………………………… 41 2.4 Materials and Methods…………………………………………………………………. 43 References………………………………………………………………………………….. 47 Chapter 3. Peptide-Glycosaminoglycan Cluster Formation Involving Cell Penetrating Peptides……………………………………………………… 50 3.1 Introduction…………………………………………………………………………….. 50 3.2 Results………………………………………………………………………………….. 53 Heparin sepharose chromatography…………………………………………….. 54 Dynamic light scattering………………………………………………………… 54 vi Cluster stability by fluorescence spectroscopy………………………………….. 55 3.3 Discussions and Conclusions…………………………………………………………… 62 3.4 Materials and methods………………………………………………………………….. 66 References………………………………………………………………………………….. 70 Chapter 4. The synthesis of thioester carbohydrate conjugates as selective lectin labelling agents............................................................ 75 4.1 Introduction…………………………………………………………………………….. 75 4.2 Results and Discussion…………………………………………………………………. 76 Synthesis of thioester constructs………………………………………………… 76 MBP labeling in vitro…………………………………………………………… 77 MBP labeling in cellular extracts and live cells………………………………… 87 4.3 Concluding remarks…………………………………………………………………….. 91 4.4 Materials and Methods…………………………………………………………………. 92 References………………………………………………………………………………….. 100 Chapter 5. Summary and Perspective…………………………………………………… 103 5.1 The importance of the spatial display of charge in peptide/protein interactions with GAGs………………………………………………………………………………….. 104 5.2 Peptide-Cluster formation involving cell penetrating peptides………………………… 105 5.3 The synthesis of thioester carbohydrate conjugates as selective lectin labelling agents.........................................................................................................
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