Jiasong Li

Significant Expansion of the Fluorescent Protein Chromophore through the Genetic Incorporation of a Metal-Chelating Unnatural Amino Acid†

Genetic incorporation of metal-binding unnatural amino acids (UAA) is a powerful method for protein sensor design, metalloenzyme engineering, and protein NMR spectroscopy. However, this method is currently underused by chemical biologists, because of the complex synthetic routes to UAAs. Herein, we report a one-step, high-yield enzymatic route for the synthesis of a novel UAA bearing an 8-hydroxyquinoline group (HqAla, Scheme 1), which forms

A Covalent Approach for Site-Specific RNA Labeling in Mammalian Cells†

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both in vitro and in mammalian cells. However, covalent RNA labeling methods with scope and versatility comparable to those of current protein labeling strategies are underdeveloped. A method is reported for the site- and sequence-specific covalent labeling of RNAs in mammalian cells by using tRNA(Ile2) -agmatidine synthetase (Tias) and click chemistry. The crystal structure of Tias in complex with an azide-bearing agmatine analogue was solved to unravel the structural basis for Tias/substrate recognition. The unique RNA sequence specificity and plastic Tias/substrate recognition enable the site-specific transfer of azide/alkyne groups to an RNA molecule of interest in vitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.

Defining the Role of Tyrosine and Rational Tuning of Oxidase Activity by Genetic Incorporation of Unnatural Tyrosine Analogs

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pKa and reduction potential in O2 reduction have not been defined despite many years of research on numerous oxidases and their models. These issues represent major challenges in our understanding of O2 reduction mechanism in bioenergetics. Through genetic incorporation of unnatural amino acid analogs of Tyr, with progressively decreasing pKa of the phenol ring and increasing reduction potential, in the active site of a functional model of oxidase in myoglobin, a linear dependence of both the O2 reduction activity and the fraction of H2O formation with the pKa of the phenol ring has been established. By using these unnatural amino acids as spectroscopic probe, we have provided conclusive evidence for the location of a Tyr radical generated during reaction with H2O2, by the distinctive hyperfine splitting patterns of the halogenated tyrosines and one of its deuterated derivatives incorporated at the 33 position of the protein. These results demonstrate for the first time that enhancing the proton donation ability of the Tyr enhances the oxidase activity, allowing the Tyr analogs to augment enzymatic activity beyond that of natural Tyr.

Significant Expansion of Fluorescent Protein Sensing Ability through the Genetic Incorporation of Superior Photo-Induced Electron-Transfer Quenchers

Photo-induced electron transfer (PET) is ubiquitous for photosynthesis and fluorescent sensor design. However, genetically coded PET sensors are underdeveloped, due to the lack of methods to site-specifically install PET probes on proteins. Here we describe a family of acid and Mn(III) turn-on fluorescent protein (FP) sensors, named iLovU, based on PET and the genetic incorporation of superior PET quenchers in the fluorescent flavoprotein iLov. Using the iLovU PET sensors, we monitored the cytoplasmic acidification process, and achieved Mn(III) fluorescence sensing for the first time. The iLovU sensors should be applicable for studying pH changes in living cells, monitoring biogentic Mn(III) in the environment, and screening for efficient manganese peroxidase, which is highly desirable for lignin degradation and biomass conversion. Our work establishes a platform for many more protein PET sensors, facilitates the de novo design of metalloenzymes harboring redox active residues, and expands our ability to probe protein conformational dynamics.

Room-temperature ferromagnetism in the Co-doped Ba0.5Sr0.5TiO3 thin films

The authors report the room-temperature ferromagnetism in the epitaxial thin films of 3% Co-doped Ba0.5Sr0.5TiO3 (CBSTO) grown by pulsed laser deposition. These films show the single phase character with Co dopants in the +2 state. More interestingly, ferromagnetic and ferroelectric transitions were observed at 570 and 150K, respectively. The CBSTO films also show the exchange bias effect manifested by the negative shift and training effect of the hysteresis loops at 5K. This work demonstrates that ferromagnetism can be induced in the ferroelectric materials, which is significant for shedding light on the mechanism of dopant induced ferromagnetism in insulators and applications.

A genetically encoded 19F NMR probe for tyrosine phosphorylation.

Tyrosine phosphorylation is a pivotal post-translational modification (PTM) which regulates enzymatic activity, protein conformation, and protein–protein interactions. While the eukaryotic protein tyrosine kinases (PTKs) have been intensively studied in the past three decades because of their great importance in cellular signaling and diseases, the prokaryotic PTKs, which play ubiquitous roles in bacterial virulence, were found only recently, and their activation mechanism is controversial. F NMR spectroscopy has recently emerged as a powerful tool for characterizing enzyme mechanisms and protein motions over a range of time scales, because of the high intrinsic sensitivity of fluorine, 100% natural abundance of the NMR-active isotope, the absence of any natural background in proteins, and the exquisite sensitivity of the F chemical shift to environment. Numerous chemical and biosynthetic methods have been developed for incorporating fluorinated amino acids into proteins. Among these methods, the genetic code expansion technique has the unique advantages that fluorinated amino acids can be directly incorporated into specific sites of any protein of interest in living cells, and that the mutant protein can be easily obtained in milligram quantities. However, this powerful method has not been applied to investigate tyrosine phosphorylation. Here we report the highly efficient genetic incorporation of the unnatural amino acid (UAA) 3,5-difluorotyrosine 1 (hereafter termed F2Y, Scheme 1), which mimics the

Interfacial potential and photoelectronic properties of manganite heterojunction La0.7Ce0.3MnO3/SrTiO3:Nb

The interfacial potential and photoelectronic properties of a heterojunction composed of La0.7Ce0.3MnO3 and SrTiO3:Nb have been experimentally studied. A two-dimensional spatial distribution of the electrostatic potential across the La0.7Ce0.3MnO3∕SrTiO3:Nb interface is obtained by the holography technique of the transmission electron microscope, which reveals the presence of a depletion layer of 8 nm in thickness at 120 K and 3 nm at 296 K and a built-in electric field within this layer. Consequently, a complex yet significant photovoltaic effect is observed. It is found that the transient photocurrent is composed of two distinctive processes with the charging-like behavior, and the time constants are surprisingly larger than that expected for a photoelectronic process, ∼30 and ∼260μs, respectively. It seems to be an intrinsic property of the manganite junction, and has nothing to do with external circuit and thermolelectric effect arising from light illumination.

Ultrafast Photoinduced Electron Transfer in Green Fluorescent Protein Bearing a Genetically Encoded Electron Acceptor

Electron transfer (ET) is widely used for driving the processes that underlie the chemistry of life. However, our abilities to probe electron transfer mechanisms in proteins and design redox enzymes are limited, due to the lack of methods to site-specifically insert electron acceptors into proteins in vivo. Here we describe the synthesis and genetic incorporation of 4-fluoro-3-nitrophenylalanine (FNO2Phe), which has similar reduction potentials to NAD(P)H and ferredoxin, the most important biological reductants. Through the genetic incorporation of FNO2Phe into green fluorescent protein (GFP) and femtosecond transient absorption measurement, we show that photoinduced electron transfer (PET) from the GFP chromophore to FNO2Phe occurs very fast (within 11 ps), which is comparable to that of the first electron transfer step in photosystem I, from P700* to A0. This genetically encoded, low-reduction potential unnatural amino acid (UAA) can significantly improve our ability to investigate electron transfer mechanisms in complex reductases and facilitate the design of miniature proteins that mimic their functions.

Rectifying and magnetotransport properties of the heterojunction of Co-doped and undoped TiO2−δ with La0.69Ca0.31MnO3 single crystal

Electron-doped wide-band-gap dilute magnetic semiconductor Ti0.93Co0.07O2−δ and TiO2−δ were grown on a hole-doped La0.69Ca0.31MnO3 single crystal to form heterojunctions. These junctions exhibit good rectifying properties and magnetoresistance effect over a relatively wide temperature range. The results for TiO2−δ were similar to that of Ti0.93Co0.07O2−δ in all respects. A schematic band structure of the junction was proposed to account for the results. This work indicates that manganite single crystals can be used as substrates for integration with other materials, which may open an alternative avenue for the exploitation of the manganite-based devices.