DECIPHERING THE PROTEOLYTIC MECHANISM OF THE ATP-DEPENDENT PROTEASE LON USING FLUORESCENT PEPTIDES by JESSICA WARD Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Thesis Adviser: Dr. Irene Lee Department of Chemistry CASE WESTERN RESERVE UNIVERSITY January, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. TABLE OF CONTENTS Title page…………………………………………………………………………………..i Committee Sign-off sheet…………………………………………………………............ii Table of Contents…………………………………………………………………………iii List of Tables…………………………………………………………………………….vii List of Figures…………………………………………………………………………...viii Acknowledgements………………………………………………………………………xii List of Abbreviations……………………………………………………………………xiii Abstract………………………………………………………………………………….xvi CHAPTER 1 Introduction to Lon protease……………..…………………………………1 CHAPTER 2 Evaluating the contributions of nucleotide binding and hydrolysis towards the proteolytic activity of E. coli Lon protease…………………………………..11 2.1 Introduction…………………………………………………………………..12 2.2 Methods and Materials……………………………………………………….15 2.2.1 Peptidase activity with various nucleotides………………………..15 2.2.2 Peptidase activity with limiting ATP……………………………....16 2.3 Results and Discussion………………………………………………………17 2.3.1 Peptidase activity with various nucleotides………………………..17 2.3.2 Peptidase activity with limiting ATP………………………………22 2.4 Conclusions…………………………………………………………………..24 CHAPTER 3 Pre-steady-state kinetic characterization of the peptidase activity of E. coli Lon protease……………………………………………………………………...28 3.1 Introduction…………………………………………………………………..29 iii 3.2 Methods and Materials……………………………………………………….32 3.2.1 Measuring peptide hydrolysis using a discontinuous acid-quench assay……………………………………………………………...32 3.2.2 Pre-steady-state kinetic analysis of peptide hydrolysis……………32 3.2.3 ADP inhibition of the lag phase in peptide hydrolysis…………….34 3.3 Results and Discussion………………………………………………………36 3.3.1 Measuring peptide hydrolysis using a discontinuous acid-quench assay……………………………………………………………..36 3.3.2 Pre-steady-state kinetic analysis of peptide hydrolysis……………37 3.3.3 ADP inhibition of the lag phase in peptide hydrolysis…………….43 3.4 Conclusions…………………………………………………………………..46 CHAPTER 4 Investigating the mechanism of E. coli Lon protease using proteolytically inactive Lon mutants……………………………………………………………..50 4.1 Introduction…………………………………………………………………..51 4.2 Methods and Materials……………………………………………………….53 4.2.1 Generation and characterization of Lon mutants…………………..53 4.2.2 Fluorescence emission scans……………………………………….54 4.2.3 Measuring peptide binding using fluorescence anisotropy………...54 4.2.4 Monitoring Lon-peptide interactions using pre-steady-state kinetic techniques………………………………………………………..55 4.2.5 ADP inhibition of the Lon-peptide interaction…………………….58 4.3 Results and Discussion………………………………………………………60 4.3.1 Developing an assay to monitor Lon-peptide interactions………...60 iv 4.3.2 Monitoring the Lon-peptide interaction with S679A using pre- steady-state kinetic techniques…………………………………69 4.3.3 Measuring peptide binding to Lon using fluorescence anisotropy.72 4.3.4 Examining events prior to peptide hydrolysis using S679W and pre-steady-state kinetic techniques…………………………73 4.3.5 ADP inhibition of the Lon-peptide interaction……………………84 4.4 Conclusions………………………………………………………………….86 CHAPTER 5 Exploring the substrate specificity of E. coli Lon protease using peptide substrates and λN protein deletion mutants……………………………………..94 5.1 Introduction………………………………………………………………….95 5.2 Materials and Methods………………………………………………………98 5.2.1 Peptide design and peptidase assays………………………………98 5.2.2 λN purification and generation of λN deletion mutants………….100 5.2.3 λN degradation assay……………………………………………..102 5.3 Results and Discussion……………………………………………………..103 5.3.1 Examining Lon substrate specificity using λN peptides…………103 5.3.2 Alanine scan of λN89-98 peptide………………………………...109 5.3.3 Importance of the C-S cleavage site in the λN89-98 peptide……114 5.3.4 Degradation of a 2-site peptide…………………………………..116 5.3.5 Wild type λN and λN deletion mutant protein purification and degradation……………………………………………………..118 5.4 Conclusions…………………………………………………………………124 CHAPTER 6 Conclusions and future directions……………………………………….127 v Appendix: Jessica Ward’s publications………………………………………………..137 Bibliography……………………………………………………………………………183 vi LIST OF TABLES CHAPTER 2 2.1. Steady-state kinetic parameters of NTP-dependent peptidase activity……………20 2.2 Peptidase activity rate constants at limiting, stoichiometric and saturating nucleotide concentrations…………………………………………………………………..23 2.3 Steady-state kinetic parameters of intrinsic and peptide stimulated NTPase activity…………………………………………………………………25 CHAPTER 3 3.1 Kinetic parameters associated with the pre-steady-state characterization of E. coli Lon protease……………………………………………………………………..41 CHAPTER 4 4.1 Primers used in site-directed mutagenesis…………………………………………..53 4.2 Kinetic parameters for ATP binding and hydrolysis by wildtype and Lon mutants...62 4.3 Kinetic constants for λN89-98 dansyl peptide interacting with S679A and S679W..72 4.4 Experimental and theoretical rate constants for the E. coli Lon mechanism………...93 CHAPTER 5 5.1 Steady-state kinetic parameters for peptidase activity with λN peptides…………..107 5.2 Steady-state kinetic parameters for ATPase activity with λN peptides……………109 5.3 Steady-state kinetic parameters for peptidase activity with λN89-98 alanine scan peptides……………………………………………………………………113 vii LIST OF FIGURES CHAPTER 1 1.1 The domain organization of E. coli Lon protease……………………………………..4 1.2 Proposed Ser-Lys dyad mechanism for peptide bond hydrolysis……………………..5 1.3 Proposed mechanism for E. coli Lon protease………………………………………...6 1.4 E. coli Lon protease mechanism proposed by our lab………………………………...7 1.5 Structures of the fluorescent and non-fluorescent model peptide substrate λN89-98…………………………………………………………………………..9 1.6 Peptidase activity assay………………………………………………………………10 CHAPTER 2 2.1 Chemical structures of nucleotides…………………………………………………..14 2.2 Scheme for ATP and peptide binding to Lon………………………………………..16 2.3 Peptidase activity at varying concentrations of peptide in the presence of saturating ATP, CTP, GTP and UTP…………………………………………….18 2.4 Peptidase activity with saturating peptide at varying concentrations of ATP, CTP, GTP and UTP………………………………………………………..19 2.5 Structures of adenine and cytidine…………………………………………………...21 2.6 Peptidase activity with limiting amounts of ATP or AMPPNP……………………...23 2.7 Limited tryptic digestion of Lon with various nucleotides…………………………..26 2.8 Proposed mechanism for peptide hydrolysis………………………………………...27 CHAPTER 3 3.1 Diagram of stopped-flow and rapid chemical quench……………………………….31 3.2 Monitoring the peptidase reaction using a discontinuous acid-quench assay……….37 viii 3.3 Stopped-flow time courses of peptide hydrolysis by E. coli Lon……………………38 3.4 Steady-state kinetics of peptide cleavage……………………………………………40 3.5 Substrate dependency of the pre-steady-state lag phase in the peptidase reaction…..42 3.6 ADP lengthens the pre-steady-state lag phase of the peptidase reaction…………….44 3.7 ADP inhibits the pre-steady-state of peptide hydrolysis……………………………..45 3.8 Pre-steady-state time courses for ATP hydrolysis and λN89-98 degradation under identical reaction conditions………………………………………………47 3.9 Proposed mechanism for peptide hydrolysis by Lon………………………………...48 CHAPTER 4 4.1 ATPase activity of Lon mutants……………………………………………………..61 4.2 Peptidase activity of Lon mutants……………………………………………………62 4.3 MANT-ATP binding to Lon…………………………………………………………63 4.4 Limited tryptic digestion analysis of S679A, S679W and S679W, W297F, W303F, W603F Lon mutants…………………………………………………...64 4.5 Emission scan of S679A Lon with the λN89-98 NF peptide……………………….65 4.6 Emission scan of S679A Lon with λN89-98 dansyl peptide………………………..66 4.7 Peptide binding to S679A can be monitored using the λN89-98 dansyl peptide and monitoring dansyl fluorescence…………………………………………….68 4.8 Peptide binding to S679A can be monitored using the λN89-98 dansyl peptide and monitoring tryptophan fluorescence…………………………………………….68 4.9 λN89-98 dansyl peptide binding to S679A is dependent on peptide……………….70 4.10 Scheme for peptide binding to Lon………………………………………………..70 4.11 λN89-98 dansyl peptide binding to S679A is dependent on ATP…………………71 ix 4.12 Equilibrium λN89-98 dansyl binding to Lon can be monitored using fluorescence anisotropy………………………………………………………………………..73 4.13 Intrinsic tryptophan fluorescence can be used to measure a conformational change in S679W dependent on ATP and AMPPNP…………………………………….75 4.14 Representative time courses for S679W interacting with the λN89-98 dansyl peptide at varying concentrations of ATP……………………………………….76 4.15 Representative time courses for S679W interacting with the λN89-98 dansyl peptide at varying concentrations of λN89-98 dansyl peptide…………………..78 4.16 The first phase of the S679W reaction is dependent on ATP………………………79