ANALYZING AND CLASSIFYING BIMOLECULAR INTERACTIONS: I. EFFECTS OF METAL BINDING ON AN IRON-SULFUR CLUSTER SCAFFOLD PROTEIN II. AUTOMATIC ANNOTATION OF RNA-PROTEIN INTERACTIONS FOR NDB Poorna Roy A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2017 Committee: Neocles Leontis, Committee Co-Chair Andrew Torelli, Committee Co-Chair Vipaporn Phuntumart, Graduate Faculty Representative H. Peter Lu © 2017 Poorna Roy All Rights Reserved iii ABSTRACT Neocles B. Leontis and Andrew T. Torelli, Committee co-chairs This dissertation comprises two distinct parts; however the different research agendas are thematically linked by their complementary approaches to investigate the nature of important intermolecular interactions. The first part is the study of interactions between an iron-sulfur cluster scaffold protein, IscU, and different transition metal ions. Interactions between IscU and specific metal ions are investigated and compared with those of SufU, a homologous Fe-S cluster biosynthesis protein from Gram-positive bacteria whose metal-dependent conformational behavior remains unclear. These studies were extended with additional metal ions selected to determine whether coordination geometry at the active sites of IscU and its homolog influence metal ion selectivity. Comparing the conformational behavior and affinity for different transition metal ions revealed that metal-dependent conformational transitions exhibited by IscU may be a recurring strategy exhibited by U-type proteins involved in Fe-S cluster biosynthesis. The second part of the thesis focuses on automated detection and annotation of specific interactions between nucleotides and amino acid residues in RNA-protein complexes. RNA- protein interactions play crucial roles in all stages of transcription, translation and gene regulation. In order to systematically detect, annotate, and query these non-covalent interactions, we have developed programs that are integrated into the RNA.BGSU.EDU data pipeline to provide RNA-protein interaction annotations to the NDB website. Our programs were then used to identify RNA-protein interactions in the mammalian mitochondrial (mmt) ribosome. Mmt ribosomes have evolved from ancestral bacterial ribosomes by large-scale reduction of ribosomal RNAs (rRNAs), loss of guanosine (G) nucleotides, and an increased prevalence of ribosomal iv proteins. Systematic comparisons of recently solved structures of small-subunits (SSU) mmt- ribosomes with those of bacteria, and of high-quality rRNA sequence alignments, allowed us to deduce rules for folding a complex RNA with far fewer Gs. Via specific RNA-protein interactions, mmt rProteins (i) substitute for truncated rRNA helices, (ii) maintain the mutual spatial orientations of the remaining helices, (iii) compensate for lost RNA-RNA interactions, (iv) reduce the solvent accessibility of exposed bases, and (v) stabilize the RNA loop motifs lacking Gs that are conserved in bacteria. v Dedicated to my parents and my grandmother, who instilled the value of education in me. Some day I hope to do the same for my baby niece, Shubhangi. vi ACKNOWLEDGMENTS I am truly thankful to my advisors Prof. Neocles B. Leontis and Prof. Andrew T. Torelli for their continuous support and guidance during my entire Ph.D. study. They not only guided my dissertation projects, but also helped me build up the skills of critical thinking. I would also like to thank my committee members, Prof. H. Peter Lu, and Prof. Vipaporn Phuntumart, for their valuable suggestions throughout my studies, and our collaborators, Prof. Craig Zirbel, Prof. Eric Westhof and Prof. Marie Sissler for their generous help and support. I am grateful to my lab mates Blake Sweeney, Maryam, Sri, Mary, Hayfa, Geetha and Mike for making my PhD work such a great learning experience. I have spent most of my PhD days, in a windowless office with Blake and Maryam, but you both made the atmosphere exciting. I am thankful to my family, dada, bourani, KD, Puntai, papa, mummy, bhaiya, bhabhi, Pihu, for keeping me healthy and sane during this challenging period. Whenever I have had doubts, my brother has led me not just by words, but also by example, showing what hard work and determination can achieve. My Kgp family has always been a constant source of support as have been the two goofballs, Ani and Shilu. My BG friends, Arpan, Sarasij, Nibedita, Kaustav, thank you for the constant encouragement. My soul sister, Debarati, for listening to my rants during frustrating times and cherishing my accomplishments. Last but not the least, my husband Vishal. You are my strength, my support, and my voice of reason. I am pretty sure you were not as worried about getting your PhD as you were about mine. We did it! vii TABLE OF CONTENTS Page CHAPTER 1. OVERVIEW OF STRUCTURAL HETEROGENITY IN Fe-S CLUSTER BIOSYNTHESIS PROTEINS …………………………………………………………...... 1 1.1 Importance of Fe-S clusters as protein cofactors ……………………………… 1 1.2 Properties of Fe-S clusters …………………………………………………… .. 1 1.3 Fe-S cluster biosynthesis systems........................................................................ 3 1.4 IscU as a metamorphic protein ........................................................................... 6 1.5 Role of IscU conformational heterogeneity in Fe-S cluster biosynthesis ........... 7 1.6 Sequence elements implicated in conformational heterogeneity of IscU and SufU homologues from Gram-negative and Gram-positive bacteria ................. 9 1.7 Interactions of IscU with metal ions and their biological relevance ................... 13 1.8 Interactions of toxic metals with Fe-S biosynthetic systems .............................. 14 1.9 Specific aims and significance of the work ......................................................... 15 REFERENCES ......................................................................................................................17 CHAPTER 2. EFFECT OF TOXIC METAL STRESS ON CONFORMATIONS OF U-TYPE PROTEINS ……………………………………………………………………… 24 2.1 IscU and SufU as metalloproteins ….......................…………………………… 24 2.1.1 Fe-S clusters targeted by transition metals … …………………………… 24 2.2 Methods …...............................................................…………………………… 26 2.2.1 Gene cloning and protein overexpression construct design ……………… 26 2.2.2 Protein expression and IMAC purification ……………………………… 26 viii 2.2.3 Iron ion content determination …...................…………………………… 27 2.2.4 Zinc ion content determination … ..................…………………………… 28 2.2.5 Removal of coordinated metal ions … ...........…………………………… 28 2.2.6 Circular Dichroism (CD) experiments ….......…………………………… 29 2.2.7 Thermofluor assay … .....................................…………………………… 30 2.2.8 Steady state fluorescence measurements … ...…………………………… 30 2.3 Results ….……………………………................................................................ 31 2.3.1 Change in secondary structure in response to metal addition …………… 31 2.3.2 Probing thermal stability of the proteins with addition of metals………... 33 2.3.3 Assessment of solvent accessibility of the protein active site upon metal binding……………… ......................................................................................... 35 2.4 Discussion……………………………................................................................ 36 2.4.1 Metal ion binding by Fe-S cluster biosynthesis proteins ………………… 36 2.4.2 Preparation of proteins for metal binding studies ……………….............. 37 2.4.3 Zn2+ alters the secondary structure profile of IscU and SufU……………. 38 2.4.4 Confirming the effect of Zn2+ on IscU and SufU with Cd2+ …………….. 39 2.4.5 IscU and SufU discriminate between transition metals with different coordination geometries ……………......................................................... 41 2.4.6 Confirming the structural effect and discrimination of different transition metals…………… ...................................................................................... 43 2.4.7 Affinity of IscU and SufU for Zn2+ ions ……………................................ 44 2.5 Conclusion ...............................................................…………………………… 45 REFERENCES ..........……………………………................................................................ 47 ix APPENDIX A FIGURES…………………………… .......................................................... 51 CHAPTER 3. ROLE OF PUTATIVE LIGANDS ON THE ACTIVE SITE OF IscU…… 53 3.1 Introduction…..........................................................…………………………… 53 3.1.1 Metals associated with proteins… ..................…………………………… 53 3.1.2 Amino acids as ligands for metal ions…………. ....................................... 54 3.1.3 Coordination geometry of metals in metalloproteins…………………….. 56 3.1.4 Conservation of residues at the active site of IscU superfamily…………. 57 3.1.5 Metal coordination at active site of IscU…………………………… ........ 59 3.1.6 Role of active site residues in conformational transitions of IscU……… . 61 3.2 Methods................................................................................................................ 62 3.2.1 Gene cloning and protein overexpression construct design……………… 62 3.2.2 Site-directed mutagenesis of IscU……………… ...................................... 63 3.2.3 Protein expression and IMAC purification ……………………………… 63 3.2.4 Iron ion content determination …...................…………………………… 64 3.2.5 Zinc ion content determination … ..................……………………………