Targeting the protein folding transition state by mutation: Large scale (un)folding rate accelerations without...

Area of Science:

• Protein dynamics and biophysics
• Protein engineering and design

Background:

• Protein folding and unfolding rates are crucial for biological function and homeostasis.
• Optimizing these rates without altering native stability is a key challenge in protein engineering.
• Mutations typically affect folding and unfolding rates inversely, complicating design efforts.

Purpose of the Study:

• To engineer protein folding and unfolding rates by targeting the folding transition state (FTS).
• To investigate the role of the FTS in the stability and function of chymotrypsin inhibitor 2 (CI2).

Main Methods:

• Targeted mutation of the CI2 folding transition state (FTS).
• Analysis of folding and unfolding rates using kinetic experiments.
• Assessment of protein stability and proteolytic degradation sensitivity.

Main Results:

• A four-mutation CI2 variant selectively stabilized the FTS.
• This variant exhibited >250-fold faster folding and unfolding rates with unchanged native stability.
• The engineered CI2 lost its inhibitory activity and became more susceptible to degradation.

Conclusions:

• Protein (un)folding rates can be precisely modulated by targeting specific residues and interactions within the FTS.
• Evolution has optimized the CI2 FTS for kinetic stability, enabling its function as a protease inhibitor.
• This FTS-centric design approach offers a promising strategy for predicting and engineering protein dynamics.