D-Glucose/D-Galactose bindng protein-ANS complex: structure stability and sugar binding affinity

PEDRO D. CLOP; SABATO D'AURIA; MARIA A. PERILLO

Montevideo - Uruguay

Congreso; 6th International Conference on Biological Physics and the 5th Southern Cone Biophysics Congress; 2007

The monomeric D-Glucose/D-Galactose-Binding Protein(GGBP) was mutated to contain a single cysteine residue at position 26. This was labeled with the sulfhydryl-reactive probe 2-(4-iodoacetamidoanilino)naphthalene-6-sulfonic acid (2,4-ANS).At room temperature, glucose (Glu), galactose (Gal) and lactose (Lac) increased the fluorescence intensity (FI) of the ANS bound to GGBP in a concentration-dependent manner, with dissociation constants 1 mM, 0.4mM and 0.3mM, respectively. The binding affinity of GGBP-ANS for Gal was similar but the effect of Glu on ANS FI was  opposed to that published previously which might have been a non-especific effect induced on an already unfolded protein. In order to investigate this hypothesis we evaluated: 1) the ANS and the intrinsic (Trp) fluorescence of GGBP-ANS as a function of urea concentration (between 0 and 8M) and temperature (15-65ºC), in the presence and in the absence of 10 mM Glu as well as  2) the effect of Glu on the FI of Trp and ANS of GGBP-ANS complex at 50º.