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

The structure of LysN contains an OB-fold motif composed of a structurally conserved five-stranded beta-barrel capped by a poorly conserved alpha-heli x between strands beta 3 and beta 4. Two additional alpha-helices, unique t o 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 exc hange measurements as a function of guanidinium hydrochloride concentration , and equilibrium unfolding transitions monitored by ellipticity at 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with Delta G(u)(0) similar to 6 kca l/ mol and m similar to 3 kcal/(molM). By contrast, NMR hydrogen exchange m easurements manifest a distribution of Delta G(u)(0) and m values which ind icate that the protein can undergo subglobal unfolding. The largest Delta G (u)(0) values from hydrogen exchange are for residues in the beta-sheet of the protein. These values, which reflect complete unfolding of the protein, are between 3 and 4 kcal/mol higher than those obtained from circular dich roism or fluorescence. This discrepancy may be due to the comparison of NMR hydrogen exchange parameters measured at residue-level resolution, with sp ectrophotometric parameters that reflect an unresolved superposition of unf olding transitions of the alpha-helices and beta-strands. The largest Delta G(u)(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the Delta G(u)(0) values ob tained from circular dichroism or fluorescence. Based on the hydrogen excha nge data, however, the three alpha-helices of LysN are on average 3 kcal/mo l less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the firs t alpha-helix of the protein retains a cooperatively folded structure. Take n together with previous results on the OB-fold proteins SN and CspA, the p resent results for LysN suggest that the most conserved elements of structu re in the OB-fold motif are the most resistant to denaturation. Ln all thre e proteins, stability to denaturation correlates with sequence hydrophobici ty. (C) 1999 Academic Press.