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 manipula ted by altering conditions in solution (for example altering urea or g lycerol concentration) and by changing the amino acid sequences. Initi al studies employed a fragment of nuclease, referred to as Delta 131 D elta, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic res onance analysis of this fragment in solution revealed a modest quantit y of dynamic structure which is native-like in character. With the add ition of urea, 12 new H-N peaks appeared in the H-1-N-15 correlation s pectrum, 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 d etected by circular dichroism spectroscopy. At very high glycerol conc entrations, an increase in helical structure became apparent. These da ta in addition to previously published results suggest that: (i) a bet a-meander (strands beta 1-beta 2-beta 3) and the second alpha helix (a lpha 2) are among the most stable local structures; (ii) the five-stra nd 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 c arboxy terminal alpha helix (alpha 3) to give the native state.