Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping

Area of Science:

• Biochemistry
• Protein Science
• Biophysics

Background:

• Globular proteins exist in equilibrium with unfolded states.
• Kinetically stable proteins (KSPs) are conformationally trapped, offering potential advantages like enhanced activity and protection against misfolding.
• Quantifying kinetic stability (KS) is crucial for understanding protein function and disease.

Purpose of the Study:

• To develop a simple, accessible method for quantifying protein kinetic stability.
• To investigate the mechanism of SDS-mediated protein denaturation.
• To apply the new method to measure the kinetic stability of challenging proteins like SOD and TTR.

Main Methods:

• Incubating proteins in SDS at high temperatures over time.
• Analyzing SDS-PAGE to quantify time-dependent loss of SDS resistance.
• Correlating SDS resistance loss with unfolding rates measured by circular dichroism.
• Applying the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR).

Main Results:

• The kinetics of SDS resistance loss linearly correlated with protein unfolding rates.
• SDS denaturation involves conformational trapping limited by unfolding rates.
• The S-TraP method successfully quantified the kinetic stability of SOD and TTR.
• Calculated unfolding half-lives at 37 °C for SOD and TTR were 70 ± 37 and 18 ± 6 days, respectively.

Conclusions:

• The S-TraP method provides a robust, cost-effective, and sample-efficient way to quantify protein kinetic stability.
• This method is applicable to impure or complex samples, facilitating broader research.
• The findings enhance our understanding of protein conformational dynamics and the biological roles of kinetic stability.