Initial Studies of the Equilibrium Folding Pathway of Staphylococcal Nuclease

• Published in: Philosophical transactions of the Royal Society of London. Series B. Biological sciences 1995-04, Vol.348 (1323), p.27-34
• Main Authors: Wang, Yi, Alexandrescu, Andrei T., Shortle, David
• Format: Article
• Language: English
• Subjects: Amino acids; Biochemistry; Chemical equilibrium; Circular Dichroism; Free energy; Glycerol - chemistry; Magnetic Resonance Spectroscopy; Micrococcal Nuclease - chemistry; Models, Theoretical; Pathways, Kinetics and Intermediates; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Proteins; Solvents; Spectral correlation; Spectroscopy; Staphylococcus; Urea - chemistry
• ISSN: 0962-8436; 1471-2970
• DOI: 10.1098/rstb.1995.0042

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 $, which lacks six structural residues from the amino terminus and one structural residue from the carboxy-terminus. Nuclear magnetic resonance analysis of this fragment in solution revealed a modest quantity of dynamic structure which is native-like in character. With the addition of urea, 12 new H$_{\text{N}}$ peaks appeared in the $^{1}$H-$^{15}$N 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.