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

National Laboratory of Biomacromolecules, Center for Structural and Molecular Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People's Republic of China

Correspondence: Address reprint requests to Jinfeng Wang, Tel.: 86-10-6488-8490(O); Fax: 86-10-6487-2026; E-mail address: jfw{at}sun5.ibp.ac.cn.

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 ß-subdomain conformation of the three fragments. V-66W110- and TMAO-stabilized SNase110 produce less compact structures having a less stable "ß-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 ß-strands in the ß-barrel of the fragments. The native-like ß-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 ß-turn regions, I-18–D-21 and Y-27–Q-30, and developed by the formation of a nonlocal nucleation site at the ß-barrel region. The formation of ß-barrel and overall structure is concerted, but the level of cooperativity is different for the three 1–110 residues SNase fragments.