Quantifying protein unfolding kinetics with a high-throughput microfluidic platform

Area of Science:

• Biochemistry and Molecular Biology
• Protein Dynamics
• Biophysical Chemistry

Background:

• Proteins can transiently sample unfolded states, risking irreversible damage like proteolysis or aggregation.
• Protein kinetic stability is a key determinant of cellular protein lifetime and abundance.
• Measuring protein unfolding rates is challenging, limiting our understanding of protein stability.

Purpose of the Study:

• To develop a high-throughput method for measuring protein unfolding rate constants.
• To overcome technical limitations in assessing protein kinetic stability.
• To enable the study of subtle effects on protein folding and stability.

Main Methods:

• Development of SPARKfold (simultaneous proteolysis assay revealing kinetics of folding), a microfluidic platform.
• High-throughput expression, purification, and measurement of unfolding rate constants using native proteolysis.
• Application to 1,104 dihydrofolate reductase ortholog samples with extensive replicates.

Main Results:

• SPARKfold successfully measured unfolding rate constants for a large number of protein samples.
• Measurements demonstrated high statistical power, resolving subtle effects on kinetic stability.
• SPARKfold-derived rate constants for five constructs correlated well with traditional methods over a 150-fold range.
• The assay provided insights into the protein folding transition state via phi (φ) analysis.

Conclusions:

• SPARKfold is a powerful, high-throughput tool for measuring protein unfolding rates and kinetic stability.
• This method facilitates the study of factors influencing protein folding and stability, including mutations.
• SPARKfold has potential applications in rational protein design for enhanced kinetic stability, particularly for industrial uses.