Site-specific water dynamics drives protein stability in hydrated deep eutectic solvents

Area of Science:

• Biochemistry
• Physical Chemistry
• Materials Science

Background:

• Deep eutectic solvents (DESs) are increasingly used as biocatalytic media due to their favorable properties.
• A detailed understanding of protein behavior and stability within DESs is lacking.
• Associated water plays a key role in protein structure and function.

Purpose of the Study:

• To investigate the impact of associated water dynamics on the thermal stability of human serum albumin (HSA) in DESs.
• To elucidate the mechanistic basis of protein stabilization in hydrated DESs.
• To explore entropy-enthalpy compensation in protein stability within DESs.

Main Methods:

• Site-selective fluorescence labeling and red-edge excitation shift measurements.
• Circular dichroism (CD) spectroscopy.
• Analysis of entropic and enthalpic contributions to protein stability.

Main Results:

• Modulation of associated water dynamics significantly alters protein stability.
• Flexible water enhances enthalpic stabilization but causes entropic destabilization.
• Restricted water shows the opposite effect, demonstrating entropy-enthalpy compensation.
• Domain-specific stability variations were observed in HSA.

Conclusions:

• Associated water dynamics are pivotal for protein stabilization in DESs.
• Understanding hydration dynamics is essential for rational design of DES-based biocatalysis.
• Site-specific stability measurements are crucial for multidomain proteins in non-conventional media.