Exploring Surfaces and Cavities in Lipoxygenase and Other Proteins by Hyperpolarized Xenon-129 NMR

Abstract

This paper presents an exploratory study of the binding interactions of xenon with the surface of several different proteins in the solution and solid states using both conventional and hyperpolarized 129 Xe NMR. The generation of hyperpolarized 129 Xe by spin exchange optical pumping affords an enhancement by 3-4 orders of magnitude of its NMR signal. As a result, it is possible to observe Xe directly bound to the surface of micromolar quantities of lyophilized protein.The highly sensitive nature of the 129 Xe line shape and chemical shift are used as indicators for the conditions most likely to yield maximal dipolar contact between 129 Xe nuclei and nuclear spins situated on the protein. This is an intermediate step toward achieving the ultimate goal of NMR enhancement of the binding-site nuclei by polarization transfer from hyperpolarized 129 Xe. The hyperpolarized 129 Xe spectra resulting from exposure of four different proteins in the lyophilized, powdered form have been examined for evidence of binding. Each of the proteins, namely, metmyoglobin, methemoglobin, hen egg white lysozyme, and soybean lipoxygenase, yielded a distinctly different NMR line shape. With the exception of lysozyme, the proteins all possess a paramagnetic iron center which can be expected to rapidly relax the 129 Xe and produce a net shift in its resonance position if the noble gas atom occupies specific binding sites near the iron. At temperatures from 223 to 183 K, NMR signals were observed in the 0-40 ppm chemical shift range, relative to Xe in the gas phase. The signals broadened and shifted downfield as the temperature was reduced, indicating that Xe is exchanging between the gas phase and internal or external binding sites of the proteins. Additionally, conventional 129 Xe NMR studies of metmyoglobin and lipoxygenase in the solution state are presented. The temperature dependence of the chemical shift and line shape indicate exchange of Xe between adsorption sites on lipoxygenase and Xe in the solvent on the slow to intermediate exchange time scale. The NMR results are compared with N 2 , Xe, and CH 4 gas adsorption isotherms. It is found that lipoxygenase is unique among the proteins studied in possessing a relatively high affinity for gas molecules, and in addition, demonstrating the most clearly resolved adsorbed 129 Xe NMR peak in the lyophilized state.

Measurement of radon and xenon binding to a cryptophane molecular host

“…Xenon binding to myoglobin has been well characterized by NMR spectroscopy (43,44) and X-ray crystallography (45), and metmyoglobin-Xe association constants have been measured to be approximately 200 and 10 M −1 (45,46). Comparable affinities have been determined for other naturally occurring sites in hemoglobin (44), lipoxygenase (47), and lipid transfer protein (48), as well as specially designed hydrophobic cavities in T4 lysozyme (49), ribose-binding protein (50), and other examples (51,52). We showed previously that xenon binds water-soluble cryptophanes approximately 1.5-fold less avidly in human plasma than in aqueous buffer solution (14).…”

Functionalized xenon as a biosensor

“…For the initial demonstration of this technique, biotin and avidin were chosen because of their high association constant (Ϸ10 15 M Ϫ1 ) (25) and the extensive literature characterizing binding properties of modified avidin or biotin (26). The cage chosen for the prototype is a cryptophane-A molecule (21) with a polar peptide chain attached to make the cryptophane water soluble.…”

“…Laserpolarized xenon is being developed as a diagnostic agent for medical magnetic resonance imaging (9) and spectroscopy (10) and as a probe for the investigation of surfaces and cavities in porous materials and biological systems. Xenon provides information both through direct observation of its NMR spectrum (11)(12)(13)(14)(15)(16)(17) and by the transfer of its enhanced polarization to surrounding spins (18,19). In a protein solution, weak xenonprotein interactions render the chemical shift of xenon dependent on the accessible protein surface and even allow the monitoring of the protein conformation (20).…”

Development of a Functionalized Xenon Biosensor

“…[35][36][37] Recently, extensive reviews regarding the history and development of hyperpolarized xenon NMR have been published. 38,39 Biological systems studied with 129 Xe NMR include globular proteins such as myoglobin and hemoglobin, [40][41][42][43][44][45][46] membrane associated peptides such as gramicidin, 47 and lipid membranes themselves. 48 While xenon shows appreciable binding to many proteins, as evidenced by its use in making heavy atom derivatives of protein crystals for x-ray crystallography, 49 In spite of the favorable attributes of xenon interacting with proteins in solution, high-sensitivity molecular sensing is not compatible with the fast-exchange characteristic of xenon-protein interactions.…”