B doi:10.1016/S0022-2836(02)00739-8 available online at http://www.idealibrary.com on w J. Mol. Biol. (2002) 322, 425–440 Detection and Characterization of Xenon-binding Sites in Proteins by 129Xe NMR Spectroscopy Seth M. Rubin1,2, Seok-Yong Lee2,3, E. Janette Ruiz1,4 Alexander Pines1,4 and David E. Wemmer1,2,3* 1Department of Chemistry Xenon-binding sites in proteins have led to a number of applications of MC-1460, University of xenon in biochemical and structural studies. Here we further develop the California, Berkeley utility of 129Xe NMR in characterizing specific xenon–protein interactions. B84A Hildebrand Hall The sensitivity of the 129Xe chemical shift to its local environment and the Berkeley, CA 94720-1460, USA intense signals attainable by optical pumping make xenon a useful NMR reporter of its own interactions with proteins. A method for detecting 2Physical Biosciences Division specific xenon-binding interactions by analysis of 129Xe chemical shift Lawrence Berkeley National data is illustrated using the maltose binding protein (MBP) from Laboratory, 1 Cyclotron Rd Escherichia coli as an example. The crystal structure of MBP in the presence Berkeley, CA 94720, USA of 8 atm of xenon confirms the binding site determined from NMR data. 3Graduate Group in Biophysics Changes in the structure of the xenon-binding cavity upon the binding of University of California maltose by the protein can account for the sensitivity of the 129Xe chemical Berkeley, CA 94720, USA shift to MBP conformation. 129Xe NMR data for xenon in solution with a 4 number of cavity containing phage T4 lysozyme mutants show that Material Sciences Division xenon can report on cavity structure. In particular, a correlation exists Lawrence Berkeley National between cavity size and the binding-induced 129Xe chemical shift. Further Laboratory, 1 Cyclotron Rd applications of 129Xe NMR to biochemical assays, including the screening Berkeley, CA 94720, USA of proteins for xenon binding for crystallography are considered. q 2002 Elsevier Science Ltd. All rights reserved Keywords: hydrophobic cavities; ligand–protein interactions; xenon *Corresponding author binding; multiple isomorphous replacement; protein conformation assay Introduction investigators can employ specific xenon–protein interactions, there exists no simple assay for xenon The affinity of xenon for hydrophobic cavities in binding. Here we further develop the use of 129Xe macromolecular interiors1 has motivated a variety NMR to detect protein cavities that bind xenon of applications of this inert gas in biochemical and and explore using 129Xe as a reporter of cavity structural studies of proteins. Xenon has been structure. used to identify protein active sites and identify The sensitivity of the 129Xe chemical shift to its cavities that might be part of pathways by which local chemical environment14 has motivated the substrates reach active sites.2–4 Because of its small use of xenon as an NMR probe of biomolecules.15–17 size and large polarizability, xenon has been used Exploiting intense optically pumped 129Xe NMR as part of model systems in both theoretical and signals,18,19 it has been possible to probe cavities in experimental studies of ligand–protein binding.5–7 lyophilized lysozyme and lipoxygenase,20 detect A recent investigation demonstrated the ability of blood oxygenation levels,21 and identify ligand- xenon to catalyze enzymatic reactions with radical binding sites in a lipid transfer protein22 through pair intermediates.8 In addition, there has been the spin-polarization induced nuclear Overhauser increasing use of xenon for determining phases effect.23 With a functionalized cage, laser-polarized in protein X-ray crystallography by both multiple xenon was used to detect a ligand binding event.24 isomorphous replacement (MIR) and multi- The chemical shift of xenon in solution with the wavelength anomalous diffraction (MAD) tech- maltose binding protein (MBP) was shown to be niques.9–13 Despite the numerous ways sensitive to the conformation of the protein.25 Although these studies have demonstrated that 129 Abbreviations used: MIR, multiple isomorphous the Xe shift changes in different protein environ- replacement; MBP, maltose binding protein. ments, the detailed mechanism for these changes 129 E-mail address of the corresponding author: remains elusive. Here we examine Xe chemical [email protected] shift changes upon binding to hydrophobic cavities 0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved 426 Detection of Xenon Binding Sites in proteins. By correlating NMR data with crystal on the 129Xe chemical shift of particular specific structures, the influence of specific xenon–protein binding sites. We examined the effects of a single interactions on the 129Xe shift is revealed. We find interaction by comparing a values for phage T4 that the presence of a xenon-binding site can be lysozyme with a xenon-binding site created or deduced from 129Xe chemical shift data alone and blocked by mutation or competitive binding. that the shift of xenon in such a site is affected by In this case, all xenon–protein interactions remain cavity structure. The implications of these findings the same except at the cavity of interest. This for applications of xenon as a biomolecular probe approach follows the work of Tilton & Kuntz, who are considered. observed changes in the 129Xe shift in myoglobin solution upon addition of HgI23, which blocks xenon binding.16 The set of T4 lysozyme mutants 129Xe chemical shifts in protein solutions described by Matthews and co-workers is an ideal Tilton & Kuntz first described the behavior of the system for such mutation and binding inhibition 129Xe NMR signal of xenon in solution with studies.29 Cavities created by mutations of bulky myoglobin.16 They observed a single resonance, hydrophobic residues bind xenon, and the struc- reflecting fast exchange between xenon in water tures of these complexes have been determined by and xenon bound to a site in the protein that had X-ray crystallography.7 By comparing the a values been identified in previous crystal structures, and of these different proteins in the presence and characterized the chemical shift as a weighted absence of xenon inhibitors, the effects of the average of the shifts of xenon in these two environ- various cavity structures on the 129Xe chemical ments. Non-specific xenon–protein interactions shift can be elucidated. were later shown to affect the 129Xe shift in myo- globin solution,26,27 resulting in a complex pattern of up- and downfield shifts as a function of xenon and myoglobin concentrations. For all proteins Results 129 studied there has only been a single Xe reso- 129Xe chemical shifts of xenon in MBP solution nance observed, with a chemical shift reflecting fast exchange of xenon among all specific and Addition of buffer with laser-polarized xenon to non-specific binding sites.20,25,27,28 Titration of 129Xe protein solutions enables rapid acquisition of 129Xe solutions with amino acid residues, peptides, NMR spectra for chemical shift determination. and denatured proteins results in a downfield Figure 1 shows a series of spectra of 1 mM xenon shift, linear in solute concentration, that results dissolved in solution with MBP at varying concen- from non-specific interactions.28 The concen- trations. Each spectrum is from a single acquisition tration-normalized change in chemical shift, with a total time for data collection of less than 5 denoted as a and expressed in units of ppm per minutes. The xenon line width increases with mM, depends on properties of the solute such as protein concentration. This broadening has been chemical functionality, charge, and size. The a observed in other protein solutions and results values of denatured proteins increase linearly from exchange of xenon between sites in the with the number of amino acid residues in the protein (both non-specific and specific) and the primary sequence with a slope approximately solvent.16,27,28 The decrease in signal with increas- equal to 0.005 ppm mM21 per amino acid residue. ing protein concentration is due to increased With the exception of myoglobin, titrations of spin–lattice relaxation of the xenon through xenon solutions with native proteins have all protein protons, causing a loss of polarization resulted in downfield shifts in the 129Xe signal that during the time required to place the sample in are linear with increasing protein concentration.25,28 the probe for data acquisition. At these concen- Despite the fact that unfolded proteins have trations of MBP and xenon, the resonance line more surface area exposed for non-specific xenon width is modest (less than 0.2 ppm), which allows interactions, the a values of several native proteins for measurement of the chemical shift with an (e.g. bovine serum albumin and unliganded MBP) estimated error of ,0.02 ppm. As seen in the are greater than for their denatured forms. It was spectra and Figure 1(b), increasing protein concen- suggested that this discrepancy arises from specific trations shift the 129Xe resonance downfield relative binding interactions in the native proteins that to xenon in buffer; the measured slope of a ¼ 2:5 ^ contribute to the 129Xe chemical shift.28 We report 0:1 ppm mM21 is similar with that reported.25 here the structure of MBP in the presence of The 129Xe chemical shifts of 4 mM xenon in MBP xenon and confirm that, as anticipated by the a solutions are also plotted in Figure 1(b). The slope value, the protein binds xenon in an interior cavity. of the titration at 4 mM xenon ða ¼ 2:4 ^ 0:1 Â Changes in xenon affinity for this cavity upon ppm mM21Þ is similar to that at 1 mM xenon. The addition of maltose can account for the sensitivity lack of xenon concentration dependence of a indi- of the 129Xe chemical shift to the conformation of cates that only a small fraction of the xenon inter- MBP.