Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) 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-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein. Study holds ProTherm entries: 14973, 14974, 14975, 14976, 14977, 14978, 14979, 14980, 14981, 14982, 14983, 14984, 14985, 14986, 14987, 14988, 14989, 14990, 14991, 14992, 14993, 14994, 14995, 14996, 14998, 14999 Extra Details: first transition phase chitosanase; tryptophan residue; site-directed mutagenesis;,thermal unfolding; molten globule
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||53|
|Proteins||Chitosanase ; Chitosanase|
|Assays/Quantities/Protocols||Experimental Assay: ddG prot_conc:3-5 micro M, temp:43.2 C ; Experimental Assay: Tm prot_conc:3-5 micro M ; Experimental Assay: dHvH prot_conc:3-5 micro M ; Experimental Assay: ddG temp:52.2 C, prot_conc:5-8 micro M ; Experimental Assay: ddG prot_conc:5-8 micro M, temp:42.7 C ; Experimental Assay: Tm prot_conc:5-8 micro M ; Experimental Assay: dHvH prot_conc:5-8 micro M ; Derived Quantity: dTm prot_conc:3-5 micro M ; Derived Quantity: dTm prot_conc:5-8 micro M|
|Libraries||Mutations for sequence AGAGLDDPHKKEIAMELVSSAENSSLDWKAQYKYIEDIGDGRGYTGGIIGFCSGTGDMLELVQHYTDLEPGNILAKYLPALKKVNGSASHSGLGTPFTKDWATAAKDTVFQQAQNDERDRVYFDPAVSQAKADGLRALGQFAYYDAIVMHGPGNDPTSFGGIRKTAMKKARTPAQGGDETTYLNGFLDARKAAMLTEAAHDDTSRVDTEQRVFLKAGNLDLNPPLKWKTYGDPYVINS|