Rational design of a genetically encoded NMR zinc sensor.

Elucidating the biochemical roles of the essential metal ion, Zn2+, motivates detection strategies that are sensitive, selective, quantitative, and minimally invasive in living systems. Fluorescent probes have identified Zn2+ in cells but complementary approaches employing nuclear magnetic resonance (NMR) are lacking. Recent studies of maltose binding protein (MBP) using ultrasensitive 129Xe NMR spectroscopy identified a switchable salt bridge which causes slow xenon exchange and elicits strong hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) NMR contrast. To engineer the first genetically encoded, NMR-active sensor for Zn2+, we converted the MBP salt bridge into a Zn2+ binding site, while preserving the specific xenon binding cavity. The zinc sensor (ZS) at only 1 µM achieved 'turn-on' detection of Zn2+ with pronounced hyper-CEST contrast. This made it possible to determine different Zn2+ levels in a biological fluid via hyper-CEST. ZS was responsive to low-micromolar Zn2+, only modestly responsive to Cu2+, and nonresponsive to other biologically important metal ions, according to hyper-CEST NMR spectroscopy and isothermal titration calorimetry (ITC). Protein X-ray crystallography confirmed the identity of the bound Zn2+ ion using anomalous scattering: Zn2+ was coordinated with two histidine side chains and three water molecules. Penta-coordinate Zn2+ forms a hydrogen-bond-mediated gate that controls the Xe exchange rate. Metal ion binding affinity, 129Xe NMR chemical shift, and exchange rate are tunable parameters via protein engineering, which highlights the potential to develop proteins as selective metal ion sensors for NMR spectroscopy and imaging.

Cryptophane-xenon complexes for 129Xe MRI applications.

The use of magnetic resonance imaging (MRI) and spectroscopy (MRS) in the clinical setting enables the acquisition of valuable anatomical information in a rapid, non-invasive fashion. However, MRI applications for identifying disease-related biomarkers are limited due to low sensitivity at clinical magnetic field strengths. The development of hyperpolarized (hp) 129Xe MRI/MRS techniques as complements to traditional 1H-based imaging has been a burgeoning area of research over the past two decades. Pioneering experiments have shown that hp 129Xe can be encapsulated within host molecules to generate ultrasensitive biosensors. In particular, xenon has high affinity for cryptophanes, which are small organic cages that can be functionalized with affinity tags, fluorophores, solubilizing groups, and other moieties to identify biomedically relevant analytes. Cryptophane sensors designed for proteins, metal ions, nucleic acids, pH, and temperature have achieved nanomolar-to-femtomolar limits of detection via a combination of 129Xe hyperpolarization and chemical exchange saturation transfer (CEST) techniques. This review aims to summarize the development of cryptophane biosensors for 129Xe MRI applications, while highlighting innovative biosensor designs and the consequent enhancements in detection sensitivity, which will be invaluable in expanding the scope of 129Xe MRI.

Monomeric Cryptophane with Record-High Xe Affinity Gives Insights into Aggregation-Dependent Sensing.

Cryptophane host molecules provide ultrasensitive contrast agents for 129Xe NMR/MRI. To investigate key features of cryptophane-Xe sensing behavior, we designed a novel water-soluble cryptophane with a pendant hydrophobic adamantyl moiety, which has good affinity for a model receptor, beta-cyclodextrin (ß-CD). Adamantyl-functionalized cryptophane-A (AFCA) was synthesized and characterized for Xe affinity, 129Xe NMR signal, and aggregation state at varying AFCA and ß-CD concentrations. The Xe-AFCA association constant was determined by fluorescence quenching, KA = 114,000 ± 5000 M-1 at 293 K, which is the highest reported affinity for a cryptophane host in phosphate-buffered saline (pH 7.2). No hyperpolarized (hp) 129Xe NMR peak corresponding to AFCA-bound Xe was directly observed at high (100 µM) AFCA concentration, where small cryptophane aggregates were observed, and was only detected at low (15 µM) AFCA concentration, where the sensor remained fully monomeric in solution. Additionally, we observed no change in the chemical shift of AFCA-encapsulated 129Xe after ß-CD binding to the adamantyl moiety and a concomitant lack of change in the size distribution of the complex, suggesting that a change in the aggregation state is necessary to elicit a 129Xe NMR chemical shift in cryptophane-based sensing. These results aid in further elucidating the recently discovered aggregation phenomenon, highlight limitations of cryptophane-based Xe sensing, and offer insights into the design of monomeric, high-affinity Xe sensors.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

Detecting protein-protein interactions by Xe-129 NMR.

Detection of protein-protein interactions (PPIs) is limited by current bioanalytical methods. A protein complementation assay (PCA), split TEM-1 ß-lactamase, interacts with xenon at the interface of the TEM-1 fragments. Reconstitution of TEM-1-promoted here by cFos/cJun leucine zipper interaction-gives rise to sensitive 129Xe NMR signal in bacterial cells.

Paramagnetic Shifts and Guest Exchange Kinetics in ConFe4-n Metal-Organic Capsules.

We investigate the magnetic resonance properties and exchange kinetics of guest molecules in a series of hetero-bimetallic capsules, [ConFe4-nL6]4- (n = 1-3), where L2- = 4,4'-bis[(2-pyridinylmethylene)amino]-[1,1'-biphenyl]-2,2'-disulfonate. H bond networks between capsule sulfonates and guanidinium cations promote the crystallization of [ConFe4-nL6]4-. The following four isostructural crystals are reported: two guest-free forms, (C(NH2)3)4[Co1.8Fe2.2L6]·69H2O (1) and (C(NH2)3)4[Co2.7Fe1.3L6]·73H2O (2), and two Xe- and CFCl3-encapsulated forms, (C(NH2)3)4[(Xe)0.8Co1.8Fe2.2L6]·69H2O (3) and (C(NH2)3)4[(CFCl3)Co2.0Fe2.0L6]·73H2O (4), respectively. Structural analyses reveal that Xe induces negligible structural changes in 3, while the angles between neighboring phenyl groups expand by ca. 3° to accommodate the much larger guest, CFCl3, in 4. These guest-encapsulated [ConFe4-nL6]4- molecules reveal 129Xe and 19F chemical shift changes of ca. -22 and -10 ppm at 298 K, respectively, per substitution of low-spin FeII by high-spin CoII. Likewise, the temperature dependence of the 129Xe and 19F NMR resonances increases by 0.1 and 0.06 ppm/K, respectively, with each additional paramagnetic CoII center. The optimal temperature for hyperpolarized (hp) 129Xe chemical exchange saturation transfer (hyper-CEST) with [ConFe4-nL6]4- capsules was found to be inversely proportional to the number of CoII centers, n, which is consistent with the Xe chemical exchange accelerating as the portals expand. The systematic study was facilitated by the tunability of the [M4L6]4- capsules, further highlighting these metal-organic systems for developing responsive sensors with highly shifted 129Xe resonances.

Ultrasound Responsive Noble Gas Microbubbles for Applications in Image-Guided Gas Delivery.

Noble gases, especially xenon (Xe), have been shown to have antiapoptotic effects in treating hypoxia ischemia related injuries. Currently, in vivo gas delivery is systemic and performed through inhalation, leading to reduced efficacy at the injury site. This report provides a first demonstration of the encapsulation of pure Xe, Ar, or He in phospholipid-coated sub-10 µm microbubbles, without the necessity of stabilizing perfluorocarbon additives. Optimization of shell compositions and preparation techniques show that distearoylphosphatidylcholine (DSPC) with DSPE-PEG5000 can produce stable microbubbles upon shaking, while dibehenoylphosphatidylcholine (DBPC) blended with either DSPE-PEG2000 or DSPE-PEG5000 produces a high yield of microbubbles via a sonication/centrifugation method. Xe and Ar concentrations released into the microbubble suspension headspace are measured using GC-MS, while Xe released directly in solution is detected by the fluorescence quenching of a Xe-sensitive cryptophane molecule. Bubble production is found to be amenable to scale-up while maintaining their size distribution and stability. Excellent ultrasound contrast is observed in a phantom for several minutes under physiological conditions, while an intravenous administration of a bolus of pure Xe microbubbles provides significant contrast in a mouse in pre- and post-lung settings (heart and kidney, respectively), paving the way for image-guided, localized gas delivery for theranostic applications.

Paramagnetic Organocobalt Capsule Revealing Xenon Host-Guest Chemistry.

We investigated Xe binding in a previously reported paramagnetic metal-organic tetrahedral capsule, [Co4L6]4-, where L2- = 4,4'-bis[(2-pyridinylmethylene)amino][1,1'-biphenyl]-2,2'-disulfonate. The Xe-inclusion complex, [XeCo4L6]4-, was confirmed by 1H NMR spectroscopy to be the dominant species in aqueous solution saturated with Xe gas. The measured Xe dissociation rate in [XeCo4L6]4-, koff = 4.45(5) × 102 s-1, was at least 40 times greater than that in the analogous [XeFe4L6]4- complex, highlighting the capability of metal-ligand interactions to tune the capsule size and guest permeability. The rapid exchange of 129Xe nuclei in [XeCo4L6]4- produced significant hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST) NMR signal at 298 K, detected at a concentration of [XeCo4L6]4- as low as 100 pM, with presaturation at -89 ppm, which was referenced to solvated 129Xe in H2O. The saturation offset was highly temperature-dependent with a slope of -0.41(3) ppm/K, which is attributed to hyperfine interactions between the encapsulated 129Xe nucleus and electron spins on the four CoII centers. As such, [XeCo4L6]4- represents the first example of a paramagnetic hyper-CEST (paraHYPERCEST) sensor. Remarkably, the hyper-CEST 129Xe NMR resonance for [XeCo4L6]4- (δ = -89 ppm) was shifted 105 ppm upfield from the diamagnetic analogue [XeFe4L6]4- (δ = +16 ppm). The Xe inclusion complex was further characterized in the crystal structure of (C(NH2)3)4[Xe0.7Co4L6]·75 H2O (1). Hydrogen bonding between capsule-linker sulfonate groups and exogenous guanidinium cations, (C(NH2)3)+, stabilized capsule-capsule interactions in the solid state and also assisted in trapping a Xe atom (∼42 Å3) in the large (135 Å3) cavity of 1. Magnetic susceptibility measurements confirmed the presence of four noninteracting, magnetically anisotropic high-spin CoII centers in 1. Furthermore, [Co4L6]4- was found to be stable toward aggregation and oxidation, and the CEST performance of [XeCo4L6]4- was unaffected by biological macromolecules in H2O. These results recommend metal-organic capsules for fundamental investigations of Xe host-guest chemistry as well as applications with highly sensitive 129Xe-based sensors.

A Structural Basis for 129 Xe Hyper-CEST Signal in TEM-1 ß-Lactamase.

Genetically encoded (GE) contrast agents detectable by magnetic resonance imaging (MRI) enable non-invasive visualization of gene expression and cell proliferation at virtually unlimited penetration depths. Using hyperpolarized 129 Xe in combination with chemical exchange saturation transfer, an MR contrast approach known as hyper-CEST, enables ultrasensitive protein detection and biomolecular imaging. GE MRI contrast agents developed to date include nanoscale proteinaceous gas vesicles as well as the monomeric bacterial proteins TEM-1 ß-lactamase (bla) and maltose binding protein (MBP). To improve understanding of hyper-CEST NMR with proteins, structural and computational studies were performed to further characterize the Xe-bla interaction. X-ray crystallography validated the location of a high-occupancy Xe binding site predicted by MD simulations, and mutagenesis experiments confirmed this Xe site as the origin of the observed CEST contrast. Structural studies and MD simulations with representative bla mutants offered additional insight regarding the relationship between local protein structure and CEST contrast.

Cryptophane Nanoscale Assemblies Expand 129Xe NMR Biosensing.

Cryptophane-based biosensors are promising agents for the ultrasensitive detection of biomedically relevant targets via 129Xe NMR. Dynamic light scattering revealed that cryptophanes form water-soluble aggregates tens to hundreds of nanometers in size. Acridine orange fluorescence quenching assays allowed quantitation of the aggregation state, with critical concentrations ranging from 200 nM to 600 nM, depending on the cryptophane species in solution. The addition of excess carbonic anhydrase (CA) protein target to a benzenesulfonamide-functionalized cryptophane biosensor (C8B) led to C8B disaggregation and produced the expected 1:1 C8B-CA complex. C8B showed higher affinity at 298 K for the cytoplasmic isozyme CAII than the extracellular CAXII isozyme, which is a biomarker of cancer. Using hyper-CEST NMR, we explored the role of stoichiometry in detecting these two isozymes. Under CA-saturating conditions, we observed that isozyme CAII produces a larger 129Xe NMR chemical shift change (δ = 5.9 ppm, relative to free biosensor) than CAXII (δ = 2.7 ppm), which indicates the strong potential for isozyme-specific detection. However, stoichiometry-dependent chemical shift data indicated that biosensor disaggregation contributes to the observed 129Xe NMR chemical shift change that is normally assigned to biosensor-target binding. Finally, we determined that monomeric cryptophane solutions improve hyper-CEST saturation contrast, which enables ultrasensitive detection of biosensor-protein complexes. These insights into cryptophane-solution behavior support further development of xenon biosensors, but will require reinterpretation of the data previously obtained for many water-soluble cryptophanes.

Xenon-Protein Interactions: Characterization by X-Ray Crystallography and Hyper-CEST NMR.

The physiological activity of xenon has long been recognized, though the exact nature of its interactions with biomolecules remains poorly understood. Xe is an inert noble gas, but can act as a general anesthetic, most likely by binding internal hydrophobic cavities within proteins. Understanding Xe-protein interactions, therefore, can provide crucial insight regarding the mechanism of Xe anesthesia and potentially other general anesthetic agents. Historically, Xe-protein interactions have been studied primarily through X-ray crystallography and nuclear magnetic resonance (NMR). In this chapter, we first describe our methods for preparing Xe derivatives of protein crystals and identifying Xe-binding sites. Second, we detail our procedure for 129Xe hyper-CEST NMR spectroscopy, a versatile NMR technique well suited for characterizing the weak, transient nature of Xe-protein interactions.

A cryptophane-based "turn-on" 129Xe NMR biosensor for monitoring calmodulin.

We present the first cryptophane-based "turn-on" 129Xe NMR biosensor, employing a peptide-functionalized cryptophane to monitor the activation of calmodulin (CaM) protein in solution. In the absence of CaM binding, interaction between the peptide and cryptophane completely suppresses the hyperpolarized 129Xe-cryptophane NMR signal. Biosensor binding to Ca2+-activated CaM produces the expected 129Xe-cryptophane NMR signal.