A strategic fluorescence labeling of D-galactose/D-glucose-binding protein from Escherichia coli helps to shed light on the protein structural stability and dynamics.
The D-glucose/D-galactose-binding protein (GGBP) of Escherichia coli serves as an initial component for both chemotaxis toward D-galactose and D-glucose and high-affinity active transport of the two sugars. GGBP is a monomer with a molecular weight of about 32 kDa that binds glucose with micromolar affinity. The sugar-binding site is located in the cleft between the two lobes of the bilobate protein. In this work, the local and global structural features of GGBP were investigated by a strategic fluorescence labeling procedure and spectroscopic methodologies. A mutant form of GGBP containing the amino acid substitution Met to Cys at position 182 was realized and fluorescently labeled to probe the effect of glucose binding on the local and overall structural organization of the protein. The labeling of the N-terminus with a fluorescence probe as well as the protein intrinsic fluorescence were also used to obtain a complete picture of the GGBP structure and dynamics. Our results showed that the binding of glucose to GGBP resulted in no stabilizing effect on the N-terminus portion of GGBP and in a moderate stabilization of the protein matrix in the vicinity of the ligand-binding site. On the contrary, it was observed that the binding of glucose has a strong stabilization effect on the C-terminal domain of the GGBP structure.
Scognamiglio
Viviana
V
Institute of Protein Biochemistry, National Research Council of Italy (CNR), Via P. Castellino, 111 80131, Naples, Italy.
Scirè
Andrea
A
Aurilia
Vincenzo
V
Staiano
Maria
M
Crescenzo
Roberta
R
Palmucci
Cristina
C
Bertoli
Enrico
E
Rossi
Mosè
M
Tanfani
Fabio
F
D'Auria
Sabato
S
D015374:Biosensing Techniques; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005947:Glucose / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D008715:Methionine / Q000737:chemistry; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D012486:Salmonella typhimurium / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D009004:Monosaccharide Transport Proteins; C509064:mglB protein, E coli; D008715:Methionine; D005947:Glucose; D003545:Cysteine