Do soft anions promote protein denaturation through binding interactions? A case study using ribonuclease A.

Area of Science:

• Biochemistry
• Chemical Physics

Background:

• Large soft anions (bromide, iodide, thiocyanate) are known protein denaturants.
• The precise mechanism by which these anions denature proteins remains unclear.

Purpose of the Study:

• Investigate the protein-denaturing properties of soft anions.
• Elucidate the mechanisms of salt-induced protein unfolding using Ribonuclease A (RNase A) as a model.

Main Methods:

• Differential scanning calorimetry to measure salt-induced perturbations in protein folding free energy.
• Utilized 1,2-hexanediol micellization as a hydrophobicity scale to separate contributions.
• Developed a methodology to distinguish Coulomb, Hofmeister, and specific anion binding effects.

Main Results:

• Sodium iodide and sodium thiocyanate significantly decreased the melting temperature of RNase A.
• Identified three mechanisms of perturbation: Coulomb screening, Hofmeister effects, and specific anion binding.
• Anion binding to polypeptide backbone moieties (CH, CH2) was confirmed as the dominant factor in protein unfolding.

Conclusions:

• Specific binding of soft anions to the protein backbone is the primary mechanism of protein denaturation.
• The magnitude of binding contributions significantly exceeds those of Coulomb screening and Hofmeister effects.
• Understanding these mechanisms is crucial for controlling protein stability and function.