Initial studies of the equilibrium folding pathway of staphylococcal nuclease.

Spectroscopic methods were used to examine the sequential build up of structure in the denatured state of staphylococcal nuclease. The 'free energy distance' between the native and denatured states was manipulated by altering conditions in solution (for example altering urea or glycerol concentration) and by changing the amino acid sequences. Initial studies employed a fragment of nuclease, referred to as delta 131 delta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new HN peaks appeared in the 1H-15N correlation spectrum, presumably as a result of the breakdown of residual structure involving the first three beta strands. With the addition of glycerol, there was a rapid increase in the quantity of beta sheet structure detected by circular dichroism spectroscopy. At very high glycerol concentrations, an increase in helical structure became apparent. These data in addition to previously published results suggest that: (i) a beta-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (alpha 2) are among the most stable local structures; (ii) the five-strand beta-barrel forms in a reaction which does not require the presence of several other native substructures; and (iii) the last step on the equilibrium folding pathway may be the formation and packing of the carboxy terminal alpha helix (alpha 3) to give the native state.

[1]  D. Shortle,et al.  Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study. , 1994, Biochemistry.

[2]  D. Shortle,et al.  NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease. , 1993, Structure.

[3]  Gerhard Wagner,et al.  NMR relaxation and protein mobility , 1993 .

[4]  S. N. Timasheff.  Water as ligand: preferential binding and exclusion of denaturants in protein unfolding. , 1992, Biochemistry.

[5]  D. Shortle,et al.  Mutational studies of protein structures and their stabilities , 1992, Quarterly Reviews of Biophysics.

[6]  G. Wider,et al.  Solvent suppression using a spin lock in 2D and 3D NMR spectroscopy with H2O solutions , 1989 .

[7]  D. Shortle,et al.  Kinetic and magnetic resonance studies of effects of genetic substitution of a Ca2+-liganding amino acid in staphylococcal nuclease. , 1986, Biochemistry.

[8]  D. Shortle,et al.  Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation , 1986, Proteins.

[9]  K. Gekko,et al.  Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. , 1981, Biochemistry.

[10]  L Mayne,et al.  Primary structure effects on peptide group hydrogen exchange. , 1972, Proteins.

[11]  Matthews Cr.  PATHWAYS OF PROTEIN FOLDING , 1993 .

[12]  W. C. Johnson,et al.  Secondary structure of proteins through circular dichroism spectroscopy. , 1988, Annual review of biophysics and biophysical chemistry.

[13]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.