Folding stability and cooperativity of the three forms of 1-110 residues fragment of staphylococcal nuclease

¹ Institute of Biophysics, Chinese Academy of Sciences

^* To whom correspondence should be addressed. E-mail: jfw{at}sun5.ibp.ac.cn.

Submitted on June 26, 2006
Revised on July 31, 2006
Accepted on 27 November 2006

	Abstract

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 G88W- and V66W-mutant SNase110, namely G88W110 and V66W110, 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-UV CD and intrinsic tryptophan fluorescence measurements. The tertiary structures and internal motions of the fragments were determined by NMR spectroscopy. Both G88W and V66W 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 degree of folding stability and compactness. G88W110 adopts a relatively rigid structure representing a most stable native-like -subdomain conformation of the three fragments. V66W110 and TMAO-stabilized SNase110 produce less compact structures having a less stable "-barrel" structural region. The different folding status accounts for the different backbone dynamic and urea-unfolding transition features of the three fragments. The G20I/G29I-mutant variants of the three fragments have provided the evidences that the folding status is correlated closely to the packing of the -strands in the "-barrel" of the fragments. The native-like "-barrel" structural region acts as a non-local nucleus for folding of the fragment. The tertiary folding of the three fragments is initiated by formation of the local nucleation sites at two -turn regions, I18-D21 and Y27-Q30, and developed by the formation of a non-local nucleation site at the "-barrel" region. The formation of "-barrel" and overall structure is concerted, but the level of cooperativity is different for the three 1-110 residues SNase fragments.

Key Words: 110-residue SNase fragments, folding cooperativity, folding stability, native-like conformations, relaxation, “Beta-barrel” structural region