Staphylococcal nuclease forms three different partially-folded
intermediates at low pH in the presence of low to moderate
concentration of anions, differing in the amount of secondary
structure, globularity, stability, and compactness. Although
these intermediates are monomeric at low protein concentration
(≤0.25 mg/mL), increasing concentrations of protein
result in the formation of dimers and soluble oligomers,
ultimately leading to larger insoluble aggregates. Unexpectedly,
increasing protein concentration not only led to association,
but also to increased structure of the intermediates. The
secondary structure, stability, and globularity of the
two less-ordered partially-folded intermediates (A1
and A2) were substantially increased
upon association, suggesting that aggregation induces structure.
An excellent correlation was found between degree of association
and amount of structure measured by different techniques,
including circular dichroism, fluorescence, Fourier transform
infrared spectroscopy (FTIR), and small-angle X-ray scattering.
The associated states were also substantially more stable
toward urea denaturation than the monomeric forms. A mechanism
is proposed, in which the observed association of monomeric
intermediates involves intermolecular interactions which
correspond to those found intramolecularly in normal folding
to the native state.