E. Janette Ruiz

Functionalized xenon as a biosensor

The detection of biological molecules and their interactions is a significant component of modern biomedical research. In current biosensor technologies, simultaneous detection is limited to a small number of analytes by the spectral overlap of their signals. We have developed an NMR-based xenon biosensor that capitalizes on the enhanced signal-to-noise, spectral simplicity, and chemical-shift sensitivity of laser-polarized xenon to detect specific biomolecules at the level of tens of nanomoles. We present results using xenon “functionalized” by a biotin-modified supramolecular cage to detect biotin–avidin binding. This biosensor methodology can be extended to a multiplexing assay for multiple analytes.

Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy.

Xenon-binding sites in proteins have led to a number of applications of xenon in biochemical and structural studies. Here we further develop the utility of 129Xe NMR in characterizing specific xenon-protein interactions. The sensitivity of the 129Xe chemical shift to its local environment and the intense signals attainable by optical pumping make xenon a useful NMR reporter of its own interactions with proteins. A method for detecting specific xenon-binding interactions by analysis of 129Xe chemical shift data is illustrated using the maltose binding protein (MBP) from Escherichia coli as an example. The crystal structure of MBP in the presence of 8atm of xenon confirms the binding site determined from NMR data. Changes in the structure of the xenon-binding cavity upon the binding of maltose by the protein can account for the sensitivity of the 129Xe chemical shift to MBP conformation. 129Xe NMR data for xenon in solution with a number of cavity containing phage T4 lysozyme mutants show that xenon can report on cavity structure. In particular, a correlation exists between cavity size and the binding-induced 129Xe chemical shift. Further applications of 129Xe NMR to biochemical assays, including the screening of proteins for xenon binding for crystallography are considered.

Optimization of Xenon Biosensors for Detection of Protein Interactions

Hyperpolarized 129Xe NMR spectroscopy can detect the presence of specific low‐concentration biomolecular analytes by means of a xenon biosensor that consists of a water‐soluble, targeted cryptophane‐A cage that encapsulates the xenon. In this work, we use the prototypical biotinylated xenon biosensor to determine the relationship between the molecular composition of the xenon biosensor and the characteristics of protein‐bound resonances. The effects of diastereomer overlap, dipole–dipole coupling, chemical‐shift anisotropy, xenon exchange, and biosensor conformational exchange on the protein‐bound biosensor signal were assessed. It was found that an optimal protein‐bound biosensor signal can be obtained by minimizing the number of biosensor diastereomers and using a flexible linker of appropriate length. Both the line width and sensitivity of chemical shift to protein binding of the xenon biosensor were found to be inversely proportional to linker length.

Distinguishing multiple chemotaxis Y protein conformations with laser-polarized 129Xe NMR

The chemical shift of the 129Xe NMR signal has been shown to be extremely sensitive to the local environment around the atom and has been used to follow processes such as ligand binding by bacterial periplasmic binding proteins. Here we show that the 129Xe shift can sense more subtle changes: magnesium binding, BeF3− activation, and peptide binding by the Escherichia coli chemotaxis Y protein. 1H‐15N correlation spectroscopy and X‐ray crystallography were used to identify two xenon‐binding cavities in CheY that are primarily responsible for the shift changes. One site is near the active site, and the other is near the peptide binding site.