A Genetically Encoded β-Lactamase Reporter for Ultrasensitive (129) Xe NMR in Mammalian Cells

Abstract

Molecular imaging holds considerable promise for elucidating biological processes in normal physiology as well as disease states, but requires noninvasive methods for identifying analytes at sub-micromolar concentrations. Particularly useful are genetically encoded, single-protein reporters that harness the power of molecular biology to visualize specific molecular processes, but such reporters have been conspicuously lacking for in vivo magnetic resonance imaging (MRI). Herein, we report TEM-1 β-lactamase (bla) as a single-protein reporter for hyperpolarized (HP) (129) Xe NMR, with significant saturation contrast at 0.1 μm. Xenon chemical exchange saturation transfer (CEST) interactions with the primary allosteric site in bla give rise to a unique saturation peak at 255 ppm, well removed (≈60 ppm downfield) from the (129) Xe-H2 O peak. Useful saturation contrast was also observed for bla expressed in bacterial cells and mammalian cells.

Keywords: NMR reporter genes; contrast agents; molecular imaging; protein biophysics; supramolecular chemistry.

Figure 1

a) Cartoon representation of bla showing secondary structure elements; color highlights the position of each residue along the primary structure, from red (N-terminus) to blue (C-terminus). The purple volumes indicate regions of high Xe occupancy. b) Selected snapshots from the molecular dynamics trajectory after 0.6 μs (top panel) and 1 μs (bottom panel), highlighting Xe atoms (blue spheres) occupying only the main allosteric site. The sidechains of the residues lining the pocket are represented as sticks.

Figure 2

a) Close-up of the main Xe binding site. Secondary structure elements--helices 1 and 12 and the flanking beta sheet—are colored red (N-terminus) to blue (C-terminus). Xe atoms (blue spheres) establish van der Waals interactions with many sidechains (gray sticks). b) Hyper-CEST z-spectra for wt-bla and I263A, 80 μM in pH 7.2 PBS.

Figure 3

Top: Time-dependent saturation transfer data for induced E. coli (left) and non-induced E. coli (right). Bottom: Time-dependent saturation transfer data for transfected (left) and control (right) HEK293T/17 cells. Saturation frequencies of Dsnob-shaped pulses were positioned +60 ppm and -60ppm referenced to Xe-aq peak, for on- and off-resonance. Pulse length, τ_pulse = 1.0496 ms; field strength, B_1,max = 279 μT. The number of pulses linearly increased from 0 to 6000, 12000 or 14000.

Scheme 1

Ultrasensitive detection of bla via HP ¹²⁹Xe chemical exchange saturation transfer. HP ¹²⁹Xe (green) exchanges into bla, where the unique resonance frequency can be saturated by shaped RF pulses. Saturated xenon (maroon) returns to the bulk, leading to a decrease in Xe-aq signal.