Allostery and GroEL: Exploring the Tenets of Nested Cooperativity

Despite a wealth of structural and biochemical studies on the functional cycle of the E. coli chaperonins GroEL and GroES, no model proposed to date accounts for all the effects seen experimentally by the various allosteric ligands: ATP, ADP, SP, GroES, and K+. The work in this dissertation explores the various allosteric transitions in the GroEL reaction cycle and offers a refined model for nested cooperativity…


Chapter 1: Introduction and Specific Aims
1.1 GroE Architecture
1.2 The Chaperonin Reaction Cycle
1.3 Allosteric Effects
1.4 Active versus Passive Models for Folding
1.5 Specific Aims
Chapter 2: General Methods and Experimental Procedures
2.1 Protein Concentrations
2.2 Bradford Assay
2.3 Polyacrylamide Gel Electrophoresis (PAGE)
2.4 Purification of GroEL
2.5 Purification of SR1
2.6 Purification of GroES
2.7 Purification of His-Tagged GroES
2.8 Coupled Enzyme ATPase Assay
2.9 Preparing Unfolded Substrate Proteins
2.10 Computer software
Chapter 3: Probing Allosteric Interactions with the Single Ring Variant, SR1
3.1 Introduction
3.2 Methods Specific to Chapter 3
3.2.1 Assaying GroES Release and SP Encapsulation in SR1 Using His-tagged GroES
3.2.2 Gel Filtration Using HPLC
3.2.3 Site Directed Mutagenesis
3.2.4 Oxidation of SR1IAX with Diamide
3.2.5 Gel Quantitation of the Reaction Coordinate
3.3 Results
3.3.1 Verifying the Oligomeric Structure of SR1
3.3.2 The Effect of Unfolded Substrate Protein and GroES on SR1 ATPase Activity
3.3.3 Response of SR1IAX ATPase Activity to Oxidation
3.3.4 Modeling the Effects of Oxidation in SRIAX
3.3.5 The Effect of Left Site versus Right Site Mutations
3.4 Discussion
3.4.1 Implications for the Allosteric Model
3.4.2 A New Oxidative Model from SR1IAX
3.4.3 The Nature of Inter-Ring Communication
Chapter 4: Examining the Effects of Potassium on the Allosteric Properties of GroEL: A New Equation for Nested Cooperativity
4.1 Introduction
4.2 Methods Specific to Chapter 4
4.2.1 Purification of Recombinant Rabbit Pyruvate Kinase
4.2.2 ATPase Assay Using the Cary 100 Bio UV Spectrophotometer
4.2.3 ATPase of GroEL and SR1 at Variable Potassium Concentrations
4.2.4 Fitting Data to the Nested Cooperativity Equations
4.3 Results
4.3.1 Examining the Effects of Potassium Concentration on the ATPase Activity of SR1
4.3.2 Fitting SR1 ATPase Activity to the Exclusive and Nonexclusive Binding Equations
4.3.3 Examining the Effects of Potassium on the ATPase Activity of GroEL
4.3.4 Fitting the GroEL ATPase Data to the Exclusive and Nonexclusive Binding Equations
4.4 Discussion
4.4.1 Evaluating a New Equation for Nested Cooperativity
4.4.2 A Proposal for the Role of Potassium
Chapter 5: Stopped Flow Analysis of GroES Association to the Asymmetric Complex: Looking at Inter-Ring Communication
5.1 Introduction
5.2 Methods Specific to Chapter 5
5.2.1 Purification and Labeling of Mutant Proteins
5.2.2 Stopped-flow Fluorescence Measurements
5.2.3 GroEL versus GroES Competitor Experiment
5.3 Results
5.3.1 GroES Association to the Trans Ring is Diffusion Limited
5.3.2 Comparing the Kinetics of Association and Dissociation
5.3.3 The Effects of K+on Association and Dissociation
5.3.4 Utilizing GroEL Traps to Study Symmetric Complex Formation
5.3.5 Testing for Symmetric Complexes Using a Steady State FRET Analysis
5.4 Discussion
5.4.1 GroES Association and Dissociation Are Tightly Coupled Events
5.4.2 The Effects of Potassium
5.4.3 The Evidence for Symmetric Complexes
Chapter 6: Summary and Final Discussion
Appendix: Derivation of Equations for Nested Cooperativity in GroEL

Author: Gresham, Jennifer Suzanne

Source: University of Maryland

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