Direct comparison of the structural dynamics between spontaneous and ligand-induced folding of staphylococcal nuclease.

Despite numerous studies focusing on the folding mechanism of globular proteins as well as ligand-induced folding of intrinsically disordered proteins (IDPs), a unified framework for understanding both types of folding mechanisms has remained elusive. To explore the similarities and differences in the structural dynamics of spontaneous versus ligand-dependent folding, we investigated the folding dynamics of staphylococcal nuclease (SNase) in the presence and absence of the substrate analog adenosine 3',5'-diphosphate (prAp). We employed equilibrium and kinetic measurements, using fluorescence and NMR spectroscopy, to study the folding of SNase coupled with the binding of prAp as a function of ligand and urea concentrations, including conditions favoring either conformational selection (CS; folding before binding) or induced fit (IF; binding before folding) scenarios. Our findings revealed that during ligand-induced folding under IF conditions, the N-terminal β-barrel domain is formed first, followed by the α-helical domain. In contrast, under CS conditions, the α-helical domain forms before the β-barrel domain. Additionally, the dynamics of ligand-induced folding mirrors the sequence of events encountered along the minor of the two parallel pathways governing the spontaneous folding process. Therefore, some of the apparent mechanistic differences between spontaneous versus ligand-induced folding can be attributed to the fact that interactions with a nucleotide ligand result in a shift in flux from the major to the minor folding pathway.