Formation of electrostatic interactions on the protein-folding pathway

Area of Science:

• Biochemistry
• Structural Biology
• Physical Chemistry

Background:

• Understanding protein folding pathways is crucial for deciphering protein function.
• Transient conformations and their electrostatic properties remain challenging to characterize.
• Ionization equilibria provide a novel avenue to probe these dynamic structures.

Purpose of the Study:

• To investigate the electrostatic interactions within transient protein folding conformations.
• To compare the H+ titration behavior of barnase across its folding pathway (denatured, intermediate, transition, and native states).
• To establish a method for analyzing transient structures using ionization equilibria.

Main Methods:

• Utilized H+ titration to analyze the ionization equilibria of specific protein residues.
• Compared the acid-titration behavior of four barnase conformations: denatured (D), intermediate (I), transition state (+), and native (N).
• Investigated the pH and ionic strength dependence of the folding intermediate's apparent stability.

Main Results:

• Identified strong electrostatic interactions in the major transition state, with some carboxylate groups exhibiting anomalous pKA values (<2).
• Observed that the transition state is an expanded form of the native state with a weakened, poorly hydrated core and loosened periphery.
• Found that the folding intermediate shares similarities with the transition state, though its interpretation requires careful consideration of equilibrium assumptions.

Conclusions:

• H+ titration behavior is a powerful tool for characterizing transient protein structures and electrostatic interactions during folding.
• The transition state of barnase folding exhibits significant electrostatic stabilization, consistent with an expanded native-like structure.
• The folding intermediate possesses distinct electrostatic properties that can be elucidated through pH and ionic strength dependence analysis.