Folding Stability and Cooperativity of the Three Forms of 1–110 Residues Fragment of Staphylococcal Nuclease

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Folding stability and cooperativity of the three forms of 1–110 residues fragment of staphylococcal nuclease (SNase110) have been studied by various biophysical and NMR methods. Samples of G-88W- and V-66W-mutant SNase110, namely G-88W110 and V-66W110, in aqueous solution and SNase110 in 2.0 M TMAO are adopted in this study. The unfolding transitions and folded conformations of the three SNase fragments were detected by far- and near-ultraviolet circular dichroism and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G-88W and V-66W single mutations as well as a small organic osmolyte (Trimethylamine N-oxide, TMAO) can fold the fragment into a native-like conformation. However, the tertiary structures of the three fragments exhibit different degrees of folding stability and compactness. G-88W110 adopts a relatively rigid structure representing a most stable native-like β-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable “ β-barrel” structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G-20I/G-29I-mutant variants of the three fragments have provided the evidence that the folding status is correlated closely to the packing of the β-strands in the β-barrel of the fragments. The native-like β-barrel structural region acts as a nonlocal nucleus for folding the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two β-turn regions, I-18–D-21 and Y-27–Q-30, and developed by the formation of a nonlocal nucleation site at the β-barrel region. The formation of β-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1–110 residues SNase fragments.