Folding subdomains of thioredoxin characterized by native-state hydrogen exchange

Abstract

Native-state hydrogen exchange (HX) studies, used in conjunction with NMR spectroscopy, have been carried out on Escherichia coli thioredoxin (Trx) for characterizing two folding subdomains of the protein. The backbone amide protons of only the slowest-exchanging 24 amino acid residues, of a total of 108 amino acid residues, could be followed at pH 7. The free energy of the opening event that results in an amide hydrogen exchanging with solvent (ΔG_op) was determined at each of the 24 amide hydrogen sites. The values of ΔG_op for the amide hydrogens belonging to residues in the helices α₁, α₂, and α₄ are consistent with them exchanging with the solvent only when the fully unfolded state is sampled transiently under native conditions. The denaturant-dependences of the values of ΔG_op provide very little evidence that the protein samples partially unfolded forms, lower in energy than the unfolded state. The amide hydrogens belonging to the residues in the β strands, which form the core of the protein, appear to have higher values of ΔG_op than amide hydrogens belonging to residues in the helices, suggesting that they might be more stable to exchange. This apparently higher stability to HX of the β strands might be either because they exchange out their amide hydrogens in a high energy intermediate preceding the globally unfolded state, or, more likely, because they form residual structure in the globally unfolded state. In either case, the central β strands-β ₃, β₂, and β₄-would appear to form a cooperatively folding subunit of the protein. The native-state HX methodology has made it possible to characterize the free energy landscape that Trx can sample under equilibrium native conditions.