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-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.
Institut für Physikalische Chemie, Westfälische Wilhelms-Universität, Schlossplatz 4/7, 48149, Münster, Germany.
C
C
Jatzke
H J
HJ
Hinz
U
U
Seedorf
G
G
Assmann
D002151:Calorimetry / Q000379:methods; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics*; D002942:Circular Dichroism; D005960:Glucosides / Q000737:chemistry; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D009154:Mutation; D010940:Plant Proteins; D011485:Protein Binding; D011489:Protein Denaturation; D013696:Temperature; D013816:Thermodynamics
C120639:1-O-decanoyl-glucoside; D002352:Carrier Proteins; D005960:Glucosides; D010940:Plant Proteins; C072087:sterol carrier proteins; D019791:Guanidine