Conformational stability and domain coupling in D-glucose/D-galactose binding protein from Escherichia coli

摘要

The monomeric D-glucose/D-galactose-binding protein (GGBP) from Escherichia coli (M, 33 000) is a periplasmic protein that serves as a high-affinity receptor for the active transport and chemotaxis towards both sugars. The effect of D-glucose binding on the thermal unfolding of the GGBP protein at pH 7.0 has been measured by differential scanning calorimetry (DSC), far-UV CD and intrinsic tryptophanyl residue fluorescence (Trp fluorescence). All three techniques reveal reversible, thermal transitions and a midpoint temperature (T-m) increase from 50 to 63degreesC produced by 10 MM D-glucose. Both in the absence and presence of D-glucose a single asymmetric endotherm for GGBP is observed in DSC, although each endotherm consists of two transitions about 4degreesC apart in T-m values. In the absence of D-glucose, the protein unfolding is best described by two non-ideal transitions, suggesting the presence of unfolding intermediates. In the presence of D-glucose protein, unfolding is more co-operative than in the absence of the ligand, and the experimental data are best fitted to a model that assumes two ideal (two-state) sequential transitions. Thus D-glucose binding changes the character of the GGBP protein folding/unfolding by linking the two domains such that protein unfolding becomes a cooperative, two two-state process. A K-A' value of 5.6 x 10(6) M-1 at 63degreesC for D-glucose binding is estimated from DSC results. The domain with the lower stability in DSC measurements has been identified as the C-terminal domain of GGBP from thermally induced Trp fluorescence changes.