Proton magnetic resonance imaging (MRI) is capable of achieving excellent anatomical resolution but is not sensitive enough for molecular imaging. The use of hyperpolarized 129Xe nuclei enhances the sensitivity of MRI. Molecular imaging is achieved by using Xe biosensors to bind Xe atoms in molecular cages that have been functionalized to target specific biomolecules. Synthetic molecular cages, most notably cryptophane-A, have typically been used to encapsulate Xe, but recently we have begun developing Xe biosensors that instead use protein to bind Xe. An advantage of protein-based Xe biosensors is that they can also serve as reporters of gene expression, analogous to green fluorescent protein (GFP). The development of a MRI-analog of GFP would take advantage of the unlimited penetration depth of MRI as well as its high spatial and temporal resolution. The detection limit of hyperpolarized Xe can be extended even further through the use of chemical exchange saturation transfer (CEST). In Hyper-CEST NMR, exchangeable solute-bound Xe atoms that resonate at a frequency different from bulk hyperpolarized Xe are selectively depolarized using RF irradiation. Rapid Xe exchange with solvent leads to loss of Xe polarization in bulk water. We have identified TEM-1 β-lactamase (TEM-1) as a single-protein Xe Hyper-CEST biosensor. TEM-1 is a 29 kDa monomeric enzyme possessing a cryptic allosteric site that was serendipitously identified by others via crystallography and later characterized computationally using Markov State Models. Molecular dynamics simulations suggest Xe affinity for this cryptic site. The Xe Hyper-CEST spectrum of pure TEM-1 in buffer reveals a 60 ppm chemical shift relative to Xe@H2O corresponding to Xe@TEM-1, and T1 contrast is observed at nanomolar TEM-1 concentrations. This T1 contrast is observed in vivo in E. coli and HEK293T cells expressing TEM-1, demonstrating TEM-1’s viability as a Xe biosensor in complex media. The crystal structure of TEM-1 derivatized with Xe reveals a high-occupancy Xe atom bound to TEM-1 inside the cryptic allosteric pocket as originally hypothesized. Mutagenesis studies are underway to elucidate the mechanism of Xe Hyper-CEST in TEM-1 as well as to enhance its Xe Hyper-CEST signal.

Single-protein reporter for hyperpolarized xenon magnetic resonance imaging

251st National Meeting (2016)

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