NMR Hydrogen Exchange of the OB-fold Protein LysN as a Function of Denaturant: The Most Conserved Elements of Structure are the Most Stable to Unfolding

Abstract

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded β-barrel capped by a poorly conserved α-helix between strands β3 and β4. Two additional α-helices, unique to the LysN structure, flank the N terminus of the OB-fold. The stability of LysN to unfolding has been investigated with NMR native state hydrogen exchange measurements as a function of guanidinium hydrochloride concentration, and equilibrium unfolding transitions monitored by ellipticity at 222nm and fluorescence at 350nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with ΔGu(0)∼6kcal/mol andm ∼3kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of ΔGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest ΔGu(0) values from hydrogen exchange are for residues in the β-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4kcal/mol higher than those obtained from circular dichroism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with spectrophotometric parameters that reflect an unresolved super position of unfolding transitions of the α-helices and β-strands. The largest ΔGu(0) values obtained from hydrogen exchange for the subset of residues in the α-helices of the protein, agree with the ΔGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three α-helices of LysN are on average 3kcal/mol less stable than the β-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first α-helix of the protein retains a cooperatively folded structure. Taken together with previous results on the OB-fold proteins SN and CspA, the present results for LysN suggest that the most conserved elements of structure in the OB-fold motif are the most resistant to denaturation. In all three proteins, stability to denaturation correlates with sequence hydrophobicity.