Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The folding of staphylococcal nuclease (SNase) is known to proceed via :a major intermediate in which the central OB subdomain,is folded and the C-terminal helical subdomain is disordered To identify the structural and energetic determinants of this folding free energy landscape, we have examined in detail;;using high pressure NMR, the consequences of cavity creating mutations in each of the two sub domains of an ultrastable. SNase,. Delta+PHS. The stabilizing mutations of Delta+PHS-enhanced,the population of the major folding intermediate Cavity creation in two different regions of the Delta+PHS reference protein, despite equivalent effects on global stability, had very distinct consequences on the complexity of the folding free energy landscape. The substitution in the C-terminal helix of Delta+PHS slightly suppressed the major intermediate and promoted an additional excited state involving disorder in the N-terminus, but otherwise decreased landscape heterogeneity with respect to the Delta+PHS background protein. The 192A substitution, located in the hydrophobic OB-fold core, had a much more profound effect, resulting in a significant increase in the number. of intermediate states and implicating the entire protein structure. Denaturant (GuHCl) had very subtle and specific effects on the landscape, suppressing senile states and favoring others, depending upon the mutational context These results demonstrate that disrupting interactions in a region of the protein with highly cooperative, unfrustrated folding has Very Profound effects on the roughness of the folding landscape, : whereas the effects are less pronounced for an energetically equivalent:substitution:in an already frustrated region.514795359546Agence National de la Recherche [PiriBio 09-455024]National Science Foundation [MCB-0543769, MCB-1050966, MCB-0743422]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)French Ministry of Research and Higher Education and a Fulbright International Graduate FellowshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)