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E Janette Ruiz

Publications and source records attributed to E Janette Ruiz.

8 recordsLinked to original sources
PubMed2006

Diastereomeric Xe chemical shifts in tethered cryptophane cages.

Cryptophane cages serve as host molecules to a Xe atom. Functionalization of cryptophane-A has permitted the development of Xe as a biosensor. Synthetic routes used to prepare cryptophanes result in racemic mixtures of the chiral cages. In the preparation of a tethered cryptophane-A cage for biosensor applications, some achiral and chiral substituents such as left-handed amino acids have been used. When the substituent is achiral, the NMR signal of the Xe atom in the functionalized cage in solution is a single isotropic peak, since the Xe shielding tensor components in the R and L cages differ by no more than the signs of the off-diagonal elements. Chiral substituents can split the cage-encapsulated Xe NMR signal into one or more sets of doublets, depending on the number of asymmetric centers in the substituent. We carry out quantum mechanical calculations of Xe nuclear magnetic shielding for the Xe atom at the same strategic position within an L cryptophane-A cage, under the influence of chiral potentials that represent r or l substituents outside the cage. Calculations of the Xe shielding response in the Lr and Ll diastereomeric pairs permit the prediction of the relative order of the Xe chemical shifts in solutions containing the Rl and Ll diastereomers. Where the substituent itself possesses two chiral centers, comparison of the calculated isotropic shielding responses in the Llr, Lrl, Rll, and Lrr systems, respectively, permits the prediction of the Xe spectrum of diastereomeric systems in solutions containing Llr, Rlr, Lll, and Rll systems. Assignment of the peaks observed in the experimental Xe NMR spectra is therefore possible, without having to undertake the difficult synthetic route that produces a single optically pure enantiomer.

Journal Article↗
PubMed2006

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.

Avidin↗
PubMed2005

NMR-based biosensing with optimized delivery of polarized 129Xe to solutions.

Laser-enhanced (LE) 129Xe nuclear magnetic resonance (NMR) is an exceptional tool for sensing extremely small physical and chemical changes; however, the difficult mechanics of bringing polarized xenon and samples of interest together have limited applications, particularly to biological molecules. Here we present a method for accomplishing solution 129Xe biosensing based on flow (bubbling) of LE 129Xe gas through a solution in situ in the NMR probe, with pauses for data acquisition. This overcomes fundamental limitations of conventional solution-state LE 129Xe NMR, e.g., the difficulty in transferring hydrophobic xenon into aqueous environments, and the need to handle the sample to refresh LE 129Xe after an observation pulse depletes polarization. With this new method, we gained a factor of >100 in sensitivity due to improved xenon transfer to the solution and the ability to signal average by renewing the polarized xenon. Polarized xenon in biosensors was detected at very low concentrations, </=250 nanomolar, while retaining all the usual information from NMR. This approach can be used to simultaneously detect multiple sensors with different chemical shifts and is also capable of detecting signals from opaque, heterogeneous samples, which is a unique advantage over optical methods. This general approach is adaptable for sensing minute quantities of xenon in heterogeneous in vitro samples, in miniaturized devices and should be applicable to certain in-vivo environments.

Biosensing Techniques↗
PubMed2005

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

The chemical shift of the (129)Xe 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 (129)Xe shift can sense more subtle changes: magnesium binding, BeF(3)(-) activation, and peptide binding by the Escherichia coli chemotaxis Y protein. (1)H-(15)N 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.

Bacterial Proteins↗
PubMed2004

Development of a functionalized xenon biosensor.

NMR-based biosensors that utilize laser-polarized xenon offer potential advantages beyond current sensing technologies. These advantages include the capacity to simultaneously detect multiple analytes, the applicability to in vivo spectroscopy and imaging, and the possibility of "remote" amplified detection. Here, we present a detailed NMR characterization of the binding of a biotin-derivatized caged-xenon sensor to avidin. Binding of "functionalized" xenon to avidin leads to a change in the chemical shift of the encapsulated xenon in addition to a broadening of the resonance, both of which serve as NMR markers of ligand-target interaction. A control experiment in which the biotin-binding site of avidin was blocked with native biotin showed no such spectral changes, confirming that only specific binding, rather than nonspecific contact, between avidin and functionalized xenon leads to the effects on the xenon NMR spectrum. The exchange rate of xenon (between solution and cage) and the xenon spin-lattice relaxation rate were not changed significantly upon binding. We describe two methods for enhancing the signal from functionalized xenon by exploiting the laser-polarized xenon magnetization reservoir. We also show that the xenon chemical shifts are distinct for xenon encapsulated in different diastereomeric cage molecules. This demonstrates the potential for tuning the encapsulated xenon chemical shift, which is a key requirement for being able to multiplex the biosensor.

Avidin↗
PubMed2003

Applications of laser-polarized 129Xe to biomolecular assays.

The chemical shift sensitivity and significant signal enhancement afforded by laser-polarized 129Xe have motivated the application of 129Xe NMR to biological imaging and spectroscopy. Recent research done by our group has used laser-polarized 129Xe in biomolecular assays that detect ligand-binding events and distinguish protein conformations. The successful application of unfunctionalized and functionalized 129Xe NMR to in vitro biomolecular assays suggests the potential future use of a functionalized xenon biosensor for in vivo imaging.

Humans↗
PubMed2002

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.

ATP-Binding Cassette Transporters↗