Proton nuclear magnetic resonance spectroscopy and ligand binding dynamics of the Escherichia coli L-arabinose binding protein

Abstract

The L-arabinose binding protein (ABP) from E. coli was studied by 1H NMR resonance spectroscopy. 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 D2O dialysis. On the basis of the pH dependence of their chemical shifts, 2 low-field resonances were tentatively assigned to C2 protons of 2 of the 3 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 on 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 at 6.degree. C of 1.3 .times. 10-7 and 1.9 .times. 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.