Development and Application of a High Throughput Protein Unfolding Kinetic Assay.

Area of Science:

• Biochemistry and Molecular Biology
• Protein Dynamics
• Biophysical Chemistry

Background:

• Protein folding and unfolding kinetics are crucial for understanding protein stability.
• Quantifying these rates offers key insights into the protein folding process.
• Existing methods may not be suitable for high-throughput screening of many 2-state folding proteins.

Purpose of the Study:

• To develop a simple, high-throughput protein unfolding kinetic assay using a plate reader.
• To validate the assay's applicability to 2-state folding proteins.
• To combine kinetic and equilibrium assays for comprehensive protein characterization.

Main Methods:

• Development of a high-throughput protein unfolding kinetic assay utilizing a plate reader.
• Validation using the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its mutants.
• Integration of the kinetic assay with a plate reader equilibrium assay.

Main Results:

• The developed assay accurately measures protein unfolding kinetics for 2-state folding proteins.
• Results for AbpSH3 and its mutants show excellent agreement with published data.
• Combined kinetic and equilibrium assays provide indirect folding rate estimates and enable ϕ-value analyses.

Conclusions:

• The high-throughput assay enables accurate kinetic and equilibrium measurements for protein mutant libraries.
• This method facilitates in-depth ϕ-value analyses.
• The assay is broadly applicable to studying the kinetics of the majority of 2-state folding proteins.