Probing Small Molecule Binding to Unfolded Polyprotein Based on its Elasticity and Refolding

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Unfolded proteins, crucial intermediates in protein synthesis and denaturation, interact with various cellular factors.
• Studying these interactions is difficult due to proteins predominantly existing in folded states under physiological conditions.

Purpose of the Study:

• To develop and demonstrate a novel method for probing small molecule binding to unfolded proteins.
• To quantify the effects of small molecule binding on the mechanical properties of unfolded proteins.

Main Methods:

• Utilized single-molecule mechanical unfolding of polyproteins.
• Employed sodium dodecyl sulfate (SDS) as a model small molecule.
• Quantified binding by measuring changes in protein elasticity and refolding dynamics.

Main Results:

• Successfully detected and quantified small molecule binding to mechanically unfolded polyproteins.
• Demonstrated that SDS binding alters the mechanical properties of unfolded proteins.
• Showcased the sensitivity of the assay to molecular interactions.

Conclusions:

• Developed a novel, label-free, single-molecule assay for studying interactions with unfolded proteins.
• This method has significant potential for investigating factors interacting with unfolded protein domains.
• The assay is applicable to various biological processes involving unfolded protein states.