Conformationally responsive dyes enable protein-adaptive differential scanning fluorimetry.

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Differential Scanning Fluorimetry (DSF) is a key technique for studying protein thermal unfolding.
• Current DSF applications are limited by poor compatibility between available fluorescent dyes and many proteins.
• Expanding protein compatibility is crucial for broader application of DSF in biological research.

Approach:

• Developed a protein-adaptive DSF (paDSF) platform utilizing a chemically diverse dye library, Aurora.
• Screened dye-protein compatibility across a wide range of proteins, including viral proteins from SARS-CoV-2.
• Employed high-throughput screening to identify optimal dye-protein pairings.

Key Points:

• Achieved 94% protein compatibility (66 out of 70 tested proteins) with the paDSF platform.
• Demonstrated a threefold increase in protein compatibility compared to previous DSF methods.
• Extended DSF's utility to observe complex biological processes like interdomain allostery in O-GlcNAc Transferase (OGT).

Conclusions:

• The paDSF platform dramatically enhances the scope of proteins amenable to DSF analysis.
• paDSF facilitates routine measurements of protein stability, dynamics, and ligand binding.
• This advancement opens new avenues for understanding protein function and interactions in various biological systems.