Site-specific dimensions across a highly denatured protein; a single molecule study

Area of Science:

• Protein folding and dynamics
• Biophysical chemistry
• Single-molecule biophysics

Background:

• The unfolded state of proteins is often modeled as a random coil.
• Discrepancies exist between ensemble and site-specific studies of denatured proteins.
• Understanding protein denatured states is crucial for protein folding research.

Purpose of the Study:

• To investigate whether highly denatured proteins adopt true random coil configurations.
• To measure residue-to-residue distances in denatured protein states.
• To compare protein structure in different denaturing conditions.

Main Methods:

• Single-molecule Forster resonance energy transfer (smFRET) techniques.
• Labeling of the FynSH3 domain with fluorescent dyes.
• Maximum likelihood analysis of transfer efficiency distributions.

Main Results:

• Dimensions of guanidine hydrochloride-unfolded protein generally match random coil predictions.
• Statistically significant, site-specific deviations from random coil behavior were observed.
• The size and shape of the trifluoroethanol-unfolded ensemble were indistinguishable from the guanidine hydrochloride-unfolded state.

Conclusions:

• Highly denatured proteins may exhibit subtle, site-specific deviations from ideal random coil behavior.
• These deviations could reconcile conflicting evidence on residual structure in unfolded proteins.
• The overall structure of the denatured state appears independent of the specific denaturant used.