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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain
Published on: December 12, 2017
LcrH, a class II chaperone from the type three secretion system, has a highly flexible native structure
Sunny K Singh1, Aimee L Boyle, Ewan R G Main
1School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK.
The Journal of Biological Chemistry
|December 13, 2012
Summary
The type three secretion system chaperone LcrH is a flexible, unstable dimer. This instability allows it to partially unfold, suggesting a "fly-casting" mechanism for binding large translocator proteins during bacterial infection.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- The type three secretion system (T3SS) is crucial for Gram-negative pathogen virulence.
- T3SS function relies on translocator proteins forming a pore in host membranes.
- Specialized chaperones are essential for translocator secretion and pore formation.
Purpose of the Study:
- To investigate the conformational stability and dynamics of the T3SS chaperone LcrH from Yersinia pestis.
- To understand how LcrH's structure relates to its function in transporting translocator proteins.
Main Methods:
- Mutational analysis of LcrH.
- Analytical ultracentrifugation to assess dimer stability.
- Equilibrium denaturation experiments to determine thermodynamic stability.
Main Results:
- LcrH exists as a thermodynamically unstable dimer (K(D) ≈15 μm).
- The tetratricopeptide repeat structure enables noncooperative unfolding to a dimeric intermediate.
- Further cooperative unfolding leads to a denatured monomeric state.
Conclusions:
- LcrH's conformational flexibility allows partially unfolded states under physiological conditions.
- This dynamic behavior supports a "fly-casting" mechanism for binding large translocator substrates.
- Understanding chaperone dynamics is key to T3SS mechanism and potential therapeutic targeting.
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