PMID:15032747

Citation	Piszczek, G, D'Auria, S, Staiano, M, Rossi, M and Ginsburg, A (2004) Conformational stability and domain coupling in D-glucose/D-galactose-binding protein from Escherichia coli. Biochem. J. 381:97-103
Abstract	The monomeric D-glucose/D-galactose-binding protein (GGBP) from Escherichia coli (M(r) 33000) 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 63 degrees C 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 4 degrees C 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 5.6x10(6) M(-1) at 63 degrees C 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.
Links	PubMed PMC1133766 Online version:10.1042/BJ20040232
Keywords	Calcium-Binding Proteins/chemistry; Calcium-Binding Proteins/metabolism; Calorimetry, Differential Scanning/methods; Circular Dichroism/methods; Escherichia coli/chemistry; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/metabolism; Fluorescence; Fluorometry/methods; Glucose/metabolism; Hot Temperature; Monosaccharide Transport Proteins/chemistry; Monosaccharide Transport Proteins/metabolism; Peptides/metabolism; Periplasmic Binding Proteins/chemistry; Periplasmic Binding Proteins/metabolism; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Tertiary; Thermodynamics; Tryptophan/chemistry
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