A Site-Specific Cross-Linker for Visible-Light Control of Proteins.

There is a need for photochemical tools that allow precise control of protein structure and function with visible light. We focus here on the s-tetrazine moiety, which can be installed at a specific protein site via the reaction between dichlorotetrazine and two adjacent sulfhydryl groups. Tetrazine's compact size enables structural mimicry of native amino acid linkages, such as an intramolecular salt bridge or disulfide bond. In this study, we investigated tetrazine installation in three different proteins, where it was confirmed that the cross-linking reaction is highly efficient in aqueous conditions and site-specific when two cysteines are located proximally: the S-S distance was 4-10 Å. As shown in maltose binding protein, the tetrazine cross-linker can replace an interdomain salt bridge crucial for xenon binding and serve as a visible-light photoswitch to modulate 129Xe NMR contrast. This work highlights the ease of aqueous tetrazine bioconjugation and its applications for protein photoregulation.

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.

Programming xenon diffusion in maltose-binding protein.

Protein interiors contain void space that can bind small gas molecules. Determination of gas pathways and kinetics in proteins has been an intriguing and challenging task. Here, we combined computational methods and the hyperpolarized xenon-129 chemical exchange saturation transfer (hyper-CEST) NMR technique to investigate xenon (Xe) exchange kinetics in maltose-binding protein (MBP). A salt bridge ∼9 Å from the Xe-binding site formed upon maltose binding and slowed the Xe exchange rate, leading to a hyper-CEST 129Xe signal from maltose-bound MBP. Xe dissociation occurred faster than dissociation of the salt bridge, as shown by 13C NMR spectroscopy and variable-B1 hyper-CEST experiments. "Xe flooding" molecular dynamics simulations identified a surface hydrophobic site, V23, that has good Xe binding affinity. Mutations at this site confirmed its role as a secondary exchange pathway in modulating Xe diffusion. This shows the possibility for site-specifically controlling xenon protein-solvent exchange. Analysis of the available MBP structures suggests a biological role of MBP's large hydrophobic cavity to accommodate structural changes associated with ligand binding and protein-protein interactions.

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.