The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity. Study holds ProTherm entries: 15099, 15100, 15101, 15102 Extra Details: Additive:EDTA 1mM isothermal titration calorimetry; SCP2; protein stability
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:45 p.m.
|Number of data points||10|
|Proteins||Non-specific lipid-transfer protein ; Non-specific lipid-transfer protein|
|Assays/Quantities/Protocols||Experimental Assay: Cm prot_conc:0.036 mg/mL ; Experimental Assay: m prot_conc:0.036 mg/mL ; Experimental Assay: dG_H2O prot_conc:0.036 mg/mL ; Experimental Assay: Cm prot_conc:0.05 mg/mL ; Experimental Assay: m prot_conc:0.05 mg/mL ; Experimental Assay: dG_H2O prot_conc:0.05 mg/mL ; Experimental Assay: Tm prot_conc:0.055 mg/mL ; Experimental Assay: dHvH prot_conc:0.055 mg/mL ; Experimental Assay: Tm prot_conc:0.44 mg/mL ; Experimental Assay: dHvH prot_conc:0.44 mg/mL|
|Libraries||Mutations for sequence SSASDGFKANLVFKEIEKKLEEEGEQFVKKIGGIFAFKVKDGPGGKEATWVVDVKNGKGSVLPNSDKKADCTITMADSDFLALMTGKMNPQSAFFQGKLKITGNMGLAMKLQNLQLQPGNAKL|