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Barriers to protein folding: formation of buried polar interactions is a slow step in acquisition of structure
C D Waldburger1, T Jonsson, R T Sauer
1Department of Biology, Massachuttes Institute of Technology, Cambridge, MA, 02139-4307, USA.
Abstract:
In the MYL mutant of the Arc repressor dimer, sets of partially buried salt-bridge and hydrogen-bond interactions mediated by Arg-31, Glu-36, and Arg-40 in each subunit are replaced by hydrophobic interactions between Met-31, Tyr-36, and Leu-40. The MYL refolding/dimerization reaction differs from that of wild type in being 10- to 1250-fold faster, having an earlier transition state, and depending upon viscosity but not ionic strength. Formation of the wild-type salt bridges in a hydrophobic environment clearly imposes a kinetic barrier to folding, which can be lowered by high salt concentrations. The changes in the position of the transition state and viscosity dependence can be explained if denatured monomers interact to form a partially folded dimeric intermediate, which then continues folding to form the native dimer. The second step is postulated to be rate limiting for wild type. Replacing the salt bridge with hydrophobic interactions lowers this barrier for MYL. This makes the first kinetic barrier rate limiting for MYL refolding and creates a downhill free-energy landscape in which most molecules which reach the intermediate state continue to form native dimers.
Insights
Mutating Arc repressor dimer salt bridges to hydrophobic interactions significantly accelerates refolding. This change alters the folding pathway, making the initial steps rate-limiting for the MYL mutant.
Area of Science:
- Protein folding dynamics
- Biophysical chemistry
- Molecular biology
Background:
- The Arc repressor dimer is a model system for studying protein folding and dimerization.
- Interactions like salt bridges and hydrogen bonds are crucial for protein stability and function.
- Understanding the kinetics of protein refolding provides insights into the energy landscape of folding.
Purpose of the Study:
- To investigate the impact of replacing salt bridges with hydrophobic interactions on the refolding and dimerization kinetics of the Arc repressor.
- To elucidate the refolding pathway and identify rate-limiting steps in the wild-type and MYL mutant Arc repressor.
Main Methods:
- Site-directed mutagenesis to create the MYL mutant Arc repressor.
- Kinetic experiments measuring refolding and dimerization rates under varying conditions (viscosity, ionic strength).
- Analysis of transition state positions and folding energy landscapes.
Main Results:
- The MYL mutant refolds and dimerizes 10- to 1250-fold faster than wild type.
- The MYL mutant exhibits an earlier transition state and dependence on viscosity, but not ionic strength.
- Wild-type salt bridge formation in a hydrophobic environment creates a kinetic barrier, which is reduced in the MYL mutant.
Conclusions:
- Replacing salt bridges with hydrophobic interactions lowers the kinetic barrier to Arc repressor refolding.
- The refolding pathway involves a partially folded dimeric intermediate, with the second step being rate-limiting for wild type.
- For the MYL mutant, the initial folding step becomes rate-limiting, leading to a downhill free-energy landscape for dimer formation.