Conformational stability and domain coupling in D-glucose/D-galactose-binding protein from Escherichia coli.
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.
Piszczek
Grzegorz
G
Section on Protein Chemistry, Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8012, USA. grzegorz_piszczek@nih.gov
D'Auria
Sabato
S
Staiano
Maria
M
Rossi
Mosè
M
Ginsburg
Ann
A
D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D005947:Glucose / Q000378:metabolism; D006358:Hot Temperature; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D002135:Calcium-Binding Proteins; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D033902:Periplasmic Binding Proteins; C023550:galactose-binding protein; D014364:Tryptophan; D005947:Glucose