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Coulombic Interactions in Ribonuclease A COULOMBIC INTERACTIONS IN RIBONUCLEASE A by Barbra Mindy Templer A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Biochemistry) at the UNIVERSITY OF WISCONSIN-MADISON 1998 A dissertation entitled Coulombic Interactions in Ribonuclease A submitted to the Graduate School of the University of Wisconsin-Madison in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Barbra Mindy Templer Date of Final Oral Examination: May 21, 1998 1998 Month & Year Degree to be awarded: December May August * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * • * * * * * * * * * * * * * * * * * * * * * * Approval Signal ures of Dissertation Readers: Signature, Dean of Graduate School l---[-;:-~ ,,(1~ h~~~ ~~~ ~ /tYk ,ec,~~ COULOMBIC INTERACTIONS IN RIBONUCLEASE A Barbra Mindy Templer Under the supervision of Associate Professor Ronald T. Raines At the University of Wisconsin-Madison The interactions between bovine pancreatic ribonuclease (RNase) A and its RNA substrate extend beyond the scissile P-Os' bond. Enzymic subsites interact with the bases and phosphoryl groups of the substrate. Those residues interacting with the phosphoryl groups comprise the PO, PI, and P2 subsites, with the scissile bond residing in the PI subsite. In this Dissertation, the function of the PO and P2 subsites of RNase A is characterized in detail. A new subsite, P( -1), is also described. Lys66 (PO subsite) and Lys7 and Argl0 (P2 subsite) were replaced with alanine residues. Wild-type RNase A and the K66A, K7 AJRlOA, and K7 AJRlONK66A variants were evaluated as catalysts for the cleavage of poly(cytidylic acid) and for their abilities to bind to single-stranded DNA, a substrate analog (Chapter Two). The values of kcal' Km, and kcalKm for poly(C) cleavage were affected by altering the PO and P2 subsites as were the values of Kd for RNase A-d(AUAA.) complex formation. Nucleic acid binding to wild-type RNase A is strongly dependent on NaCI concentration (values of Kd range from 0.82 f..1.M - 88 f..1.M), but this dependence is diminished upon alteration of the PO and P2 subsites. Similar experiments with NaF and wild-type RNase A indicate that the anion has little effect on nucleic acid binding. Structures ofK7AJRI0AIK66A RNase A and the K7AJRIOAIK66A· uridine 3'­ phosphate RNase A complex were determined by X-ray diffraction analyses to resolutions of 2.0 Aand 2.1 A, respectively (Chapter Three). Little change is observed between these structures and that of wild-type RNase A, either free or with bound cytidine 3'-phosphate. The II PO and P2 subsites depress the pKa values of the two active-site histidine residues and decrease their interaction with each other. These effects are sensitive to salt concentration. For these reasons, K7NRIONK66A RNase A cleaves UpA and binds uridine 3'-phosphate, neither of which span the PO or P2 subsites, less efficiently than does wild-type RNase A. In Chapter Four, a new RNase A subsite, comprised of Arg85 , is described. Together these results provide a detailed picture of the role of Coulombic interactions in catalysis by RNase A. ll1 ACKNOWLEDGEMENTS Many people have helped me throughout my years in the Raines laboratory, and to them I owe a big thank you. Jeung-Hoi Ha taught me the basics of molecular biology and protein purification and also created the plasmid encoding K66A RNase A. Chapter Two of this Dissertation evolved from work Jeung-Hoi started during her work as a post-doc in the Raines laboratory. L. Wayne Schultz guided me in the crystallization of the K7AIRIOAIK66A RNase A variant and solved the X-ray structures discussed in this dissertation. David Quirk assisted me with NMR experiments and data analysis. Juneko Grilley, an undergraduate with whom I have worked, performed many of the experiments in Chapter Four. Brad Kelemen read 99% of this Dissertation, providing excellent editing skills and valuable discussions. Tony Klink proofread all of my chapters. June Messmore, Chiwook Park, Kim Taylor, Jon Wilkins, Paula Wittmayer and Ken Woycechowsky provided excellent suggestions and helpful discussions about all of my work. All members of the Raines laboratory, past and present. provided a pleasant work environment and were excellent sources of scientific ideas. My advisor. Ronald Raines. has been an excellent mentor. He has challenged me to think about biophysics in-depth, taught me to write well and pay attention to details. and provided a supportive environment in which to earn my Ph.D. M. Thomas Record. Jr. suggested several of the experiments in this Dissertation and provided excellent insight into the interpretation of many of my results. Ruth Saecker and several other Record lab members provided helpful discussions about protein-nucleic acid interactions. John Markley and members of the NMRF AM provided many helpful suggestions about NMR experiments. Mark Anderson made my life at the 500 MHz NMR instrument much more pleasant than it could have been. Adam Fisher. my husband. and my parents are owed great thanks for believing in me. motivating me to finish, and being constant sources of support throughout graduate school. iv TABLE OF CONTENTS Abstract ..................................................................................................... i Acknowledgements ........................................................................... , .......... iii Table of Contents ......................................................................................... iv List of Figures ............................................................................................ vi List of Tables ........................................................................................... viii Abbreviations ............................................................................................. ix Chapter 1 Introduction ....................................................................................... 1 Chapter 2 Coulombic Forces in Protein - RNA Interactions: Binding and Cleavage by Ribonuclease A and Variants at Lys7, ArglO, and Lys66 ....................... 21 2.1 Abstract ..................................................................................... 22 2.2 Introduction ......... " ..... " '" ................................................. , ........ , 24 2.3 Experimental Procedures ................................................................. 27 2.4 Results ...................................................................................... 34 2.5 Discussion .................................................................................. 37 2.6 Acknowledgement .................................................................... " ... 46 Chapter 3 Coulombic Effects of Remote Subsites on the Active Site of Ribonuclease A ... '" ... 64 3.1 Abstract ..................................................................................... 65 3.2 Introduction ................................................................................ 67 3.3 Experimental Procedures ................................................................. 69 v 3.4 Results ...................................................................................... 77 3.5 Discussion .................................................................. .. '" ........... 81 3.6 Acknowledgement ......................................................................... 97 Chapter 4 A New Remote Subsite in Ribonuclease A ................................................ 125 4.1 Abstract .................................................................................... 126 4.2 Introduction .............................................................................. 127 4.3 Experimental Procedures ................................................................ 129 4.4 Results ..................................................................................... 134 4.5 Discussion .................................................................. .............. 136 4.6 Acknowledgement. ....................................................................... 140 Chapter 5 References ...................................................................................... 151 vi LIST OF FIGURES Figure 1.1 Charles Augustin Coulomb ............................................................. 15 Figure 1.2 Ribbon diagram of the structure of RNase A......................................... 17 Figure 1.3 Mechanisms of the transphosphorylation and hydrolysis reactions catalyzed by RNase A ................................................................................. 19 Figure 2.1 Structure of RNase A complexed to the substrate analog d(AT AAG) ............ 50 Figure 2.2 Schematic representation of the binding of an RNA fragment to RNase A ...... 52 Figure 2.3 Auorescein-Iabeled oligonucleotide [Fl-d(AUAA)] ................................. 54 Figure 2.4 Thermodynamic cycles for the cleavage of poly(C) by RNase A upon replacing Lys7 and Argl0, Lys66, or all three with alanine residues ......................... 56 Figure 2.5 Raw data for the binding of fluorescein-d(AUAA) to wild-type RNase A at four [Na+] ...................................................................................... 58 Figure 2.6 Transformed data for binding of fluorescein-d(AUAA) to wild-type RNase A ... ............................................................................................. 60 Figure 2.7 Transformed data for binding of fluorescein-d(AUAA) to wild-type
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