Contribution of a buried hydrogen bond to lambda repressor folding kinetics

Area of Science:

• Protein Folding Dynamics
• Biophysical Chemistry
• Molecular Biology

Background:

• Understanding protein folding mechanisms is crucial for comprehending biological function and disease.
• Specific interactions, like hydrogen bonds, play a significant role in protein stability and folding pathways.
• Monomeric lambda repressor serves as a model system for studying small helical protein folding.

Purpose of the Study:

• To investigate the contribution of a specific hydrogen bond (Asp 14-Ser 77) to the stability of monomeric lambda repressor.
• To elucidate the role of this hydrogen bond in the protein folding and unfolding pathways.
• To compare the folding behavior of wild-type lambda repressor with a faster-folding mutant.

Main Methods:

• Site-directed mutagenesis to remove the Asp 14-Ser 77 hydrogen bond by mutating residues to alanine.
• Double mutant cycle analysis to quantify the energetic contribution of the hydrogen bond.
• Kinetic experiments measuring protein folding and unfolding rates.
• Computational modeling using diffusion-collision approximations.

Main Results:

• The Asp 14-Ser 77 hydrogen bond stabilizes the native state of lambda repressor by 1.5 kcal/mol.
• Removal of this hydrogen bond primarily affects unfolding rates, with minimal impact on folding rates.
• This suggests the hydrogen bond is formed after rate-limiting steps in the folding pathway.
• Folding of helix 3 appears to be a critical slow step preceding the formation of the 14-77 hydrogen bond.

Conclusions:

• The Asp 14-Ser 77 hydrogen bond is a post-folding interaction, contributing to native state stability rather than guiding the folding process.
• Helix 3 folding is a key determinant in the folding pathway of monomeric lambda repressor.
• A combined experimental and computational strategy can effectively unravel folding mechanisms of small helical proteins.