Detection of a conformational change in maltose binding protein by (129)Xe NMR spectroscopy.

Since the observation of a xenon binding site in the interior of myoglobin,1 the interactions of proteins with xenon have been studied with the goal of using the inert gas as a biomolecular probe. Xenon is relatively small and hydrophobic, and it binds weakly (K < ∼102 M-1) to the nonpolar interiors of many proteins with little perturbation to the protein structure.2 The sensitivity of the xenon chemical shift to its local environment3 has motivated magnetic resonance studies of xenon in biological systems.4 More recently, the intense 129Xe NMR signals attainable with optical pumping techniques5 have been used to probe cavities in lyophilized lysozyme and lipoxygenase,6 detect blood oxygenation levels,7 and identify xenon binding sites in a lipid transfer protein8 by the spin-polarization induced nuclear Overhauser effect.9 Here we report the dependence of the 129Xe chemical shift on the specific native conformation of the maltose binding protein (MBP) from Escherichia coli. The ability to discriminate protein conformations through the 129Xe chemical shift indicates the potential use of 129Xe NMR for direct assessment of protein functional states and ligand binding events. MBP is a periplasmic protein in Gram-negative bacteria that plays a role in active transport and serves as an initial receptor for chemotaxis.10 MBP (MW ≈ 41 700) binds maltose, other linear maltodextrins, and cyclodextrins with high affinity, but it binds glucose with low affinity.11 The three-dimensional structures of MBP, both unligated and complexed with maltose, have been determined by X-ray crystallography to 1.8 and 2.3 Å respectively.12 The sugar binding site is located in a cleft between the two domains of the protein; maltose binding (K ≈ 9 × 105 M-1) induces a large structural change from an “open” unligated conformer (Figure 1a and 1b) to a “closed” structure of the complex (Figure 1c). The conformational change of MBP upon addition of maltose has been detected in solution by a number of physical measurements including fluorescence, electron paramagnetic resonance, small-angle X-ray scattering, and 1H NMR.11,13 Figure 1 shows 129Xe spectra of laser-polarized xenon in a 350 μM solution of MBP in the absence of ligand (a) and in the presence of 1 mM â-cyclodextrin (b) and 1 mM maltose (c).14 The 129Xe resonance is shifted by 0.35 ppm in the spectrum of xenon in solution with the closed MBP conformation. This change in the spectrum upon addition of maltose indicates that the 129Xe chemical shift is sensitive to the conformational state of MBP.15 To characterize further the effect of MBP conformation on the 129Xe chemical shift, shift measurements were made on a series of MBP solutions at varying protein concentration in the absence and presence of maltose; the results for the titrations are plotted in Figure 2. The single resonance observed in each spectrum indicates that xenon is in fast exchange between the buffer and protein interaction sites. Analogous behavior has been observed for a number of proteins.4,6,16 The observed shift is the average of the 129Xe chemical shift in each environment weighted by the occupancy of xenon in that environment. As described previously, these environments can all be treated as weak binding sites that correspond either to diffusion-mediated nonspecific interactions between xenon and the protein surface or to specific xenon binding sites in the protein.16 As observed in Figure 2, the overall chemical shift changes linearly with protein concentration in the limit where a small fraction of xenon is bound to any site. The concentration-normalized shift, R (units of ppm/mM), has a characteristic value for a protein that depends on the number, strength, and effect on the 129Xe shift of all the nonspecific and