Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein.
The L-arabinose binding protein (ABP) from Escherichia coli was studied by proton nuclear magnetic resonance spectroscopy (1H NMR). Distinct spectral changes occur when ABP binds its natural ligand, L-arabinose, which involve resonances in the aromatic ring current shifted methyl, bulk methyl, methylene, aromatic, and amide proton regions of the spectra. Several amide resonances can be "protected" from deuterium exchange if L-arabinose is bound to ABP prior to deuterium oxide dialysis. On the basis of the pH dependence of their chemical shifts, two low-field resonances have been tentatively assigned to C2 protons of two of the three histidines present in ABP. These histidyl residues have pK values of 8.0 and 8.6 which support their involvement in ionic interactions observed earlier in the crystallographic analysis. One histidyl residue shows a small chemical shift change upon the addition of arabinose. When ABP binds D-galactose, changes in the spectra occur which are different than those observed when L-arabinose is bound. Binding of L-arabinose and D-galactose to the binding protein (ABP) was considered by equilibrium binding and fluorescence emission spectroscopy. ABP binds L-arabinose and D-galactose with high affinities (Kd's at 6 degrees C of 1.3 x 10(-7) and 1.9 x 10(-7) M, respectively), and both enthalpy and entropy contribute to the ABP-ligand association. When excited at 285 nm, ABP has a fluorescence emission maximum of 340 nm which is quenched and blue shifted (to 337 nm) upon binding L-arabinose. ABP binding D-galactose produced a similar emission shift but no fluorescence quenching.
D001426:Bacterial Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
C016242:AraF protein, E coli; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D005690:Galactose