Native-like Partially Folded Conformations and Folding Process Revealed in the N-terminal Large Fragments of Staphylococcal Nuclease: A Study by NMR Spectroscopy

The N-terminal large fragments of staphylococcal nuclease (SNase), SNase110 (1–110 residues), SNase121 (1–121 residues), and SNase135 (1–135 residues), and the fragment mutants G88W110, G88W121, V66W110 and V66W121 were studied by heteronuclear multidimensional NMR spectroscopy. Ensembles of co-existent native-like partially folded and unfolded states were observed for fragments. The persistent native-like tertiary interaction drives fragments to be in partially folded states, which reveal native-like β-barrel conformations. G88W and V66W mutations modulate the extent of inherent native-like tertiary interaction in fragment molecules, and in consequence, fragment mutants fold into native-like β-subdomain conformations. In cooperation with the inherent tertiary interaction, 2 M TMAO (trimethylamine N-oxide) can promote the folding reaction of fragments through the changes of unfolding free energy, and a native-like β-subdomain conformation is observed when the chain length contains 135 residues. Heterogeneous partially folded conformations of 1–121 and 1–135 fragments due to cis and trans X-prolyl bond of Lys116–Pro117 make a non-unique folding pathway of fragments. The folding reaction of fragments can be characterized as a hierarchical process.