A Dynamic Hydrophobic Core and Surface Salt Bridges Thermostabilize a Designed Three-Helix Bundle

Area of Science:

• Protein engineering
• Biophysics
• Computational biology

Background:

• Thermostable proteins are valuable for industrial applications, pharmaceuticals, and biosensors.
• Designing de novo thermostable proteins remains a challenge in bioengineering.
• UVF is a de novo protein with significantly higher thermostability than its parent EnHD.

Purpose of the Study:

• To elucidate the structural, dynamic, and thermodynamic basis of UVF's high thermostability.
• To investigate the independent contributions of UVF's hydrophobic core and charged surface to its stability.
• To compare the stability mechanisms of UVF, EnHD, and chimeric proteins.

Main Methods:

• 4 μs all-atom molecular dynamics simulations at varying temperatures.
• Tanford-Kirkwood electrostatic calculations.
• Thermodynamic analysis using weighted-histogram analysis method on structure-based models.

Main Results:

• UVF's hydrophobic core promotes dynamics, reducing entropy loss upon folding.
• UVF's charged surface provides favorable electrostatic interactions, contributing enthalpically to stability.
• Both the hydrophobic core and charged surface independently enhanced thermostability in chimeric proteins.

Conclusions:

• The combination of a dynamic hydrophobic core and a charged surface drives UVF's exceptional thermostability.
• Understanding these features allows for the intentional design of novel thermostable proteins.
• This work provides a framework for engineering proteins with tailored stability for specific applications.