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Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry
Published on: June 7, 2018
Differential Interactions between Tat-specific redox enzyme peptides and their chaperones
Catherine S Chan1, Limei Chang, Kenton L Rommens
1Department of Biological Sciences, University of Calgary, Alberta, Canada.
Journal of Bacteriology
|January 20, 2009
Summary
The twin-arginine translocase (Tat) system utilizes specific chaperones (REMPs) to mature redox enzymes. This study reveals REMPs can bind unrelated enzymes, with interactions preventing degradation and enhancing binding affinity.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- The twin-arginine translocase (Tat) system transports prefolded proteins across the bacterial cytoplasmic membrane.
- Tat substrates are characterized by an N-terminal SRRxFLK twin-arginine (RR) motif.
- Redox enzyme maturation proteins (REMPs) act as specific chaperones for many Tat substrates in Escherichia coli.
Purpose of the Study:
- To characterize the interactions between 10 REMPs and 15 RR peptides from Tat-specific redox enzyme subunits.
- To investigate the specificity and functional implications of REMP-RR peptide interactions.
- To identify factors influencing REMP-substrate recognition and binding.
Main Methods:
- In vitro and in vivo binding assays.
- Biacore surface plasmon resonance (SPR) for kinetic analysis.
- Bacterial two-hybrid and far-Western experiments.
- Sequence alignments and analysis of full-length protein sequences.
Main Results:
- Some REMPs exhibited specificity for functionally similar redox enzymes, while others showed broader recognition.
- SPR and bacterial two-hybrid identified interactions not detected by far-Western assays, suggesting roles for conformational freedom or cellular factors.
- REMP-RR interactions protected both partners from proteolytic degradation in vivo.
- The presence of multiple binding partners led to up to 10-fold tighter binding to the expected RR substrate.
- Homology between REMPs and the hydrophobic regions C-terminal to the RR motif correlated with cross-recognition.
Conclusions:
- REMP specificity is not absolute and can extend to enzymes of diverse functions.
- Protein-protein interactions within the Tat pathway are dynamic and influenced by cellular context.
- REMP binding is crucial for stabilizing Tat substrates and enhancing their maturation efficiency.
- Sequence homology provides a basis for understanding cross-recognition patterns between REMPs and RR peptides.
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