Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp(28), Trp (101), and Trp(227)) were mutated to phenylalanine, and thermal unfolding e xperiments of the proteins were done in order to investigate the role of tr yptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expressi on system using Streptomyces lividans TK24. Each unfolding curve obtained b y CD at 222 mm did not exhibit a two-state transition profile, but exhibite d a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint tem perature (T-m) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-typ e. On the other hand, each unfolding curve obtained by tryptophan fluoresce nce exhibited a typical two-state profile and agreed with the first phase o f transition curves obtained by CD. Differential scanning calorimetry profi les of the proteins were consistent with the data obtained by CD. These dat a suggested that the mutation of individual tryptophan residues would partl y collapse the side chain interactions, consequently decreasing T-m and enh ancing the formation of a molten globule-like intermediate in the thermal u nfolding process. The tryptophan side chains are most likely to play import ant roles in cooperative stabilization of the protein. (C) 1999 Elsevier Sc ience B.V. All rights reserved.