Interactions between subdomains in the partially folded state of staphylococcal nuclease

摘要

Staphylococcal nuclease can be roughly divided into a β-subdomain in N-terminal and an α-subdomain in C-terminal. They fold sequentially under certain conditions, causing a partially folded intermediate state in which the native-like β-barrel persists while α-helix regions largely disorder. To investigate the possible long-range interactions between the two subdomains in the intermediate, N-terminal fragments have been used as intermediate analogues, with polypeptide ending at positions 102, 110, 121 and 135 and with a tryptophan substitution at position 66 or 88 to facilitate the observation of the β-barrel. Segment-resolved interactions between β-barrel and residues 103-135 were identified by comparing their spectroscopic properties of fluorescence, circular dichroism and NMR and by their stability. Except for unstable V66W102, the guanidine and thermal denaturation of fragments are cooperative and well approximated by the two-state transition. Minimal stable structure units of both tryptophan-containing fragments comprise residues 1-110. With the main interaction in segment 103-135, residues 103-110 contribute approximate 2 kcal/mol to the stability. Elongation of C-terminal from 110 residue neither increases the stability nor alters the structure core of the G88W fragments. However, residues 111-121 influence the tertiary structure of the V66W fragments suggesting its minor interactions with β-barrel.