Allostery in GroEL: Its Role in the Refolding of Protein Substrates

The Escherichia coli chaperonin GroEL assists in the re-folding of misfolded substrate proteins (SPs). In response to the binding of ATP, GroEL undergoes large, allosteric structural transitions, resulting in an expansion of its central cavity and a capping of the cavity by the co-chaperonin GroES. Bound SP is released into the central cavity following the structural transitions…


Chapter 1: Introduction and Specific Aims
1.1 The Structure of GroEL
1.2 Why Does Nature Need GroEL?
1.3 The Workings of GroEL
1.4 The GroE Reaction Cycle
1.5 Active or Passive Refolding?
1.6 Allosteric Domain Movements Drive Unfolding
1.7 Specific Aims
Chapter 2: General Methods and Experimental Procedures
2.1 A Note on Protein Concentration
2.2 Site Directed Mutagenesis of GroEL and GroES
2.3 Polyacrylamide Gel Electrophoresis (PAGE)
2.4 Purification of GroEL
2.5 Purification of GroES
2.6 Purification of His-Tagged GroES
2.7 Preparing GroEL Cysteine Mutants for Cross-Linking or Labeling
2.8 GroEL Cross-linking
2.9 Coupled-enzyme ATPase Assay
2.10 Preparing Unfolded Protein Substrates
2.11 Computer Software
Chapter 3: Characterization of a GroEL Intersubunit Double Cysteine Mutant: R197C / E386C
3.1 Introduction
3.2 Methods Specific to Chapter 3
3.2.1 Mutagenesis
3.2.2 Gradient Gels
3.2.3 Purification of Phosphate Binding Protein
3.2.4 Labeling of PBP
3.2.5 ATPase Assay Using PBP
3.2.6 Assaying GroES Release From the EL/ES Bullet Using 14-ATP
3.2.7 Assaying GroES Release From the EL/ES Bullet Using His-tagged GroES
3.3 Data Analysis
3.3.1 Gel Quantitation of the Reaction Coordinate
3.3.2 Developing Models that Predict GroELIRX’s Response to Cross-Linking
3.4 Results
3.4.1 Cross-linking of GroELIRX
3.4.2 Native Cysteines are Non-reactive
3.4.3 Kinetics of Diamide Oxidation and DTT Reduction Demonstrate that Oxidation is Stochastic
3.4.4 GroELIRX Has Reduced Overall Cooperativity Compared to GroELWT
3.4.5 Locking GroELIRX Into the TT State Eliminates Cooperativity
3.4.6 Two Cross-links Are Needed in a GroELIRX Ring to Lock it in the T State
3.4.7 One Cross-link Per GroELIRX Ring is Sufficient to Prevent GroES Binding
3.4.8 GroELIRX’s Ability to Release GroES is Compromised
3.4.9 Unfolded SP Does Not Stimulate GroELIRX ATPase Actvity
3.5 Discussion
Chapter 4: Allosteric Basis for the Actions of SP on GroEL ATPase Activity: Evidence for Active Unfolding
4.1 Introduction
4.2 Methods Specific to Chapter 4
4.2.1 Assay for MDH Activity During Re-folding by GroEL
4.3 Results
4.3.1 Effect of SPs on GroEL ATPase Activity
4.3.2 Titration of Unfolded SP
4.3.3 Refolding of MDH by GroEL/GroES
4.3.4 X-linked GroELIAX is not Affected by SP
4.3.5 Adding Unfolded SP to GroELIAX Provides a Direct Measurement of VmaxT
4.3.6 SP is not a Perfect Mimic of a Cross-link
4.3.7 Short Peptides and a Hydrophobic Amino Acid do not Stimulate ATPase Activity
4.4 Discussion
Chapter 5: Examining the Allosteric Basis for the Release of GroES from the GroEL/GroES Complex
5.1 Introduction
5.2 Methods Specific to Chapter 5
5.2.1 Mutagenesis and Purification
5.2.2 Labeling GroEL E315C and GroES 98C with Fluorescent Probes
5.2.3 Stopped-Flow Fluorescence Measurements
5.2.4 Measuring the Kinetics of GroES Release Using GroESHis
5.3 Results
5.3.1 Confirming the Presence of FRET in the Experimental System
5.3.2 Measuring GroES Release Using FRET
5.3.3 GroES Dissociation Results Differ…


Author: Grason, John Peter

Source: University of Maryland

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