title: 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 beta-barrel capped by a poorly conserved alpha-helix between strands beta3 and beta4. Two additional alpha-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 222 nm and fluorescence at 350 nm. The spectrophotometric measurements suggest an apparent two-state unfolding transition with DeltaGu(0) approximately 6 kcal/mol and m approximately 3 kcal/(molM). By contrast, NMR hydrogen exchange measurements manifest a distribution of DeltaGu(0) and m values which indicate that the protein can undergo subglobal unfolding. The largest DeltaGu(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 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 alpha-helices and beta-strands. The largest DeltaGu(0) values obtained from hydrogen exchange for the subset of residues in the alpha-helices of the protein, agree with the DeltaGu(0) values obtained from circular dichroism or fluorescence. Based on the hydrogen exchange data, however, the three alpha-helices of LysN are on average 3 kcal/mol less stable than the beta-sheet. Consistent with the subglobal unfolding of LysN evinced by hydrogen exchange, a deletion mutant that lacks the first alpha-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.
authors.affiliation: Department of Structural Biology, Biozentrum, University of Basel, Basel, CH-4056, Switzerland. alexandrescu@ubaclu.unibas.ch
authors.forename: A T
authors.initials: AT
authors.lastname: Alexandrescu
authors.forename: V A
authors.initials: VA
authors.lastname: Jaravine
authors.forename: S A
authors.initials: SA
authors.lastname: Dames
authors.forename: F P
authors.initials: FP
authors.lastname: Lamour
mesh_terms: D000926:Anticodon / Q000378:metabolism; D017124:Conserved Sequence; D019791:Guanidine; D006859:Hydrogen; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D009841:Oligonucleotides; D009844:Oligosaccharides; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary
chemical_list: D000926:Anticodon; D009841:Oligonucleotides; D009844:Oligosaccharides; D006859:Hydrogen; D008250:Lysine-tRNA Ligase; D019791:Guanidine