Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2

The temperature- and solvent-induced denaturation of both the SCP2 wild-typ e and the mutated protein c71s were studied by CD measurements at 222 nm. T he temperature-induced transition curves were deconvoluted according to a t wo-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 corre sponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stabili ty parameters characterizing the guanidine hydrochloride denaturation curve s were also calculated on the basis of a two-state transition. The transiti on 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 tw o proteins. A quantitative measure are the Gibbs energies Delta G(D)(0)(buf fer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for th e mutant. We characterized also the alkyl chain binding properties of the t wo proteins by measuring the interaction parameters for the complex formati on with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalori metry. The dissociation constants, K-d, for wild-type SCP2 are 335 mu M at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpi es, Delta H-b, are -21.5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degre es C. The parameters for the c71s mutant at 25 degrees C are K-d = 413 mu M and Delta H-b = 16.6 kJ/mol. These results suggest that both SCP2 wild-typ e and the c71s mutant bind the hydrophobic compound with moderate affinity. (C) 1999 Elsevier Science B.V. All rights reserved.