Investigation of the Zinc Binding Region of Prothymosin-alpha: A Spectroscopic and Thermodynamic Approach to Study Metal Binding in Intrinsically Disordered Proteins Abstract The goal of this study is to provide a deeper insight into how metals bind intrinsically disordered proteins (IDPs) by taking zinc binding to prothymosin-alpha as a case study. The involvement of metals in several diseases caused by (IDPs) has been noted long ago (Cu binding of α-synuclein in Parkinsons disease, prion aggregation due to Cu in madcow disease. etc.). However, there is still a lack of understanding of how metal binds IDPs. A deeper insight into metal binding by IDPs is essential for a full understanding of a proteome and to pave a way for drug development. Prothymosin-alpha (Protα) is an acidic, natively unfolded, and highly conserved protein located mostly in the nucleus of eukaryotic cells. It consists of 110 amino acids out of which 53 residues are aspartic (D) and glutamic (E) acids thus it is highly acidic. The exact biological role of the protein is unknown. However, it has been shown to be involved in cell proliferation, chromatin remodeling, antiapoptotic activity etc. One of the most interesting characteristics of Protα is its ability to bind metal cations like Ca2+, Mg2+, Mn2+, Zn2+, Al2+. In the background of all these cations, Protα selectively interacts with Zn2+ and undergoes a structural change plus the interactions of the protein with its binding partners are enhanced in the presence of Zn2+. These features underline the importance of Zn2+ binding in Protα. Very little information is available about Zn2+ binding to Protα. A recent NMR study has shown that the 48-110 segment of Protα is the Zn2+ binding region of the protein. Although the 48-110 region is shown to be the Zn2+ binding region, the central 50-89 segment, where most of the Zn2+ binding residues are located in the protein, was used for all the studies here. To aid in the synthesis, purification, and characterization of highly negatively charged sequences like Protα(50-89)N50W new amino acid derivatives, 4-pyridylmethyl protected glutamic and aspartic acids were developed and their application was tested. Subsequently a circular dichroism (CD) spectroscopic study on Protα(50-89)N50W revealed that the peptide, like the full length protein, undergoes a structural change in the presence of Zn2+ and both the full protein and the peptide were saturated at 75 eq of Zn2+ indicating that the majority of Zn added binds in this region. The above study had also shown that selective binding of Zn2+ by Protα(50-89)N50W is due to sequence specificity and not solely because of electrostatic interactions between Protα(50- 89)N50W and Zn2+. Further the CD and differential scanning calorimetry (DSC) studies have shown that Protα(50-89)N50W undergoes a structural change similar to an alpha helix when heated from 20-80 oC. To set the stage for determining the specific glutamic and aspartic residues in Prot that act as ligands for Zn2+, a proof of principle for spin inversion recovery experiments has been provided using Zn2+ binding to EDTA as a model. This idea can further be extended to Protα(50- 89)N50W to understand if Zn2+ binds to a specific set of residues or if several degenerate binding sites exist. Investigation of the Zinc Binding Region of Prothymosin-alpha: A Spectroscopic and Thermodynamic Approach to Study Metal Binding in Intrinsically Disordered Proteins By SriRamya Garapati A Dissertation Submitted to the Faculty of East Carolina University In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Chemistry East Carolina University, North Carolina 2014 ©SriRamya Garapati, 2014 Investigation of the Zinc Binding Region of Prothymosin-alpha: A Spectroscopic and Thermodynamic Approach to Study Metal Binding in Intrinsically Disordered Proteins By SriRamya Garapati APPROVED BY: DIRECTOR OF DISSERTATION: ______________________________________________________________________________ Colin Burns, Ph.D. COMMITTEE MEMBER: ______________________________________________________________________________ William Allen, Ph.D. COMMITTEE MEMBER: ______________________________________________________________________________ Art Rodriguez, Ph.D. COMMITTEE MEMBER: ______________________________________________________________________________ Anthony Kennedy, Ph.D. COMMITTEE MEMBER: ______________________________________________________________________________ Cindy Putnam Evans, Ph.D. DIRECTOR OF INTERDISCIPLINARY PROGRAM IN BIOLOGICAL SCIENCES: ______________________________________________________________________________ Terry West, Ph.D. DEAN OF THE GRADUATE SCHOOL: ______________________________________________________________________________ Paul Gemperline, Ph.D. DEDICATED To My beloved parents and husband Acknowledgements I heart fully thank my advisor, Dr. Colin Burns, without whom I could not have made through graduate school. His encouragement, guidance and support helped me in every way to develop an understanding of the subject. His patience and simplicity has always surprised me. I am very grateful for all his help in this journey I would like to extend my gratitude to Dr. Art Rodriguez and Dr. Anthony Kennedy who added their valuable suggestions and time to my research. I also thank my fellow graduate students for all the support they have offered throughout this journey. A special thanks to my committee members and Department of Chemistry for their assistance and time. Lastly, I offer my regards to all of those who supported me in any respect during the completion of the project. Table of Contents Dedication ......................................................................................................................................... ii Acknowledgements .......................................................................................................................... iii List of Figures .................................................................................................................................. vi List of Tables ................................................................................................................................. xiii List of Sequences ........................................................................................................................... xiv List of Schemes ............................................................................................................................... xv List of Abbreviation and Symbols ................................................................................................. xvi Chapter 1: Introduction 1.1 Natively Unfolded Proteins ........................................................................................................ 1 1.2 General Introduction About Prothymosin-α ............................................................................. 13 1.3 Biological Role of Prothymosin-α ........................................................................................... 15 1.4 Metal Binding Characteristics of Prothymosin-α .................................................................... 18 1.5 Research Goal and Overview of Research Strategy ................................................................ 26 Chapter 2: The 4-Pyridylmethyl Ester as a Protecting Group for Aspartic and Glutamic Acid: Flipping Peptide Charge States for Characterization by Positive Mode ESI-MS 2.1 Introduction .............................................................................................................................. 34 2.2 Research Strategy..................................................................................................................... 41 2.3 Synthesis, Purification and Characterization of 4-Pyridylmethyl Ester of Aspartic and Glutamic Acid ................................................................................................................................ 45 2.4 Application of 4-Pyridylmethyl Esters of Aspartic and Glutamic Acid in the Synthesis of Model Peptides................................................................................................................................ 50 2.5 Conclusions .............................................................................................................................. 57 Chapter 3: Zn and Temperature Induced Structural Changes in Protα(50-89)N50W Studied Using Circular Dichroism (CD) Spectroscopy 3.1 Introduction to CD Spectroscopy............................................................................................. 58 3.2 Research Strategy..................................................................................................................... 71 3.3 CD Measurements .................................................................................................................... 75 Chapter 4: Zn and Temperature Induced Structural Changes in Protα(50-89)N50W Studied Using Differential Scanning Calorimetry (DSC) 4.1 Introduction to DSC ................................................................................................................. 89 4.2 Research Strategy ..................................................................................................................... 99 4.3 DSC Measurements ............................................................................................................... 101 Chapter 5: Spin-Lattice Relaxation Measurements