Binding of glucose to the D-galactose/D-glucose-binding protein from Escherichia coli restores the native protein secondary structure and thermostability that are lost upon calcium depletion.
The effect of the depletion of calcium on the structure and thermal stability of the D-galactose/D-glucose-binding protein (GGBP) from Escherichia coli was studied by fluorescence spectroscopy and Fourier-transform infrared spectroscopy. The calcium-depleted protein (GGBP-Ca) was also studied in the presence of glucose (GGBP-Ca/Glc). The results show that calcium depletion has a small effect on the secondary structure of GGBP, and, in particular it affects a population of alpha-helices with a low exposure to solvent. Alternatively, glucose-binding to GGBP-Ca eliminates the effect induced by calcium depletion by restoring a secondary structure similar to that of the native protein. In addition, the infrared and fluorescence data obtained reveal that calcium depletion markedly reduces the thermal stability of GGBP. In particular, the spectroscopic experiments show that the depletion of calcium mainly affects the stability of the C-terminal domain of the protein. However, the binding of glucose restores the thermal stability of GGBP-Ca. The thermostability of GGBP and GGBP-Ca was also studied by molecular dynamics simulations. The simulation data support the spectroscopic results. New insights into the role of calcium in the thermal stability of GGBP contribute to a better understanding of the protein function and constitute important information for the development of biotechnological applications of this protein. Mutations and/or labelling of amino acid residues located in the protein C-terminal domain may affect the stability of the whole protein structure.
D'Auria
Sabato
S
Istituto di Biochimica delle Proteine, CNR, Via P. Castellino, 111 80131 Napoli, Italy. s.dauria@ibp.cnr.it
Ausili
Alessio
A
Marabotti
Anna
A
Varriale
Antonio
A
Scognamiglio
Viviana
V
Staiano
Maria
M
Bertoli
Enrico
E
Rossi
Mosè
M
Tanfani
Fabio
F
D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005947:Glucose / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013696:Temperature
D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; C509064:mglB protein, E coli; D005947:Glucose; D002118:Calcium