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1. chinaXiv:201605.01808 [pdf]

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

Liu, Xiaohong; Jiang, Li; Li, Jiasong; Wang, Li; Zhou, Qing; Lv, Xiaoxuan; Gong, Weimin; Wang, Jiangyun; Lu, Yi; Lu, Yi; Yu, Yang
Subjects: Biology >> Biophysics

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.

submitted time 2016-05-18 Hits3339Downloads1505 Comment 0

2. chinaXiv:201605.01533 [pdf]

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Taira, Kazunari; Zhang, Lihe; Zhou, Demin; Wang, Qi; Chen, Peng; Wang, Jiangyun
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

submitted time 2016-05-12 Hits10809Downloads1329 Comment 0

3. chinaXiv:201605.01531 [pdf]

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

Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Wang, Jiangyun; Lv, Xiaoxuan; Li, Jiasong; Zhou, Qing; Wang, Jiangyun; Yu, Yang; Lu, Yi; Cui, Chang; Mukherjee, Arnab; Lu, Yi; Hosseinzadeh, Parisa; Lu, Yi; Nilges, Mark J.
Subjects: Biology >> Biophysics

While a conserved tyrosine (Tyr) is found in oxidases, the roles of phenol ring pK(a) and reduction potential in O-2 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 O-2 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 O-2 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.

submitted time 2016-05-12 Hits1841Downloads991 Comment 0

4. chinaXiv:201605.01485 [pdf]

Synthetic Model of the Oxygen-Evolving Center: Photosystem II under the Spotlight

Hu, Cheng; Liu, Xiaohong; Wang, Jiangyun; Yu, Yang
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The oxygen-evolving center (OEC) in photosystem II catalyzes a water splitting reaction. Great efforts have already been made to artificially synthesize the OEC, in order to elucidate the structure-function relationship and the mechanism of the reaction. Now, a new synthetic model makes the best mimic yet of the OEC. This recent study opens up the possibility to study the mechanism of photosystem II and photosynthesis in general for applications in renewable energy and synthetic biology.

submitted time 2016-05-12 Hits1573Downloads785 Comment 0

5. chinaXiv:201605.01451 [pdf]

A Designed Metalloenzyme Achieving the Catalytic Rate of a Native Enzyme

Liu, Xiaohong; Wang, Jiangyun; Yu, Yang; Lu, Yi; Cui, Chang; Petrik, Igor D.; Lu, Yi
Subjects: Biology >> Biophysics

Terminal oxidases catalyze four-electron reduction of oxygen to water, and the energy harvested is utilized to drive the synthesis of adenosine triphosphate. While much effort has been made to design a catalyst mimicking the function of terminal oxidases, most biomimetic catalysts have much lower activity than native oxidases. Herein we report a designed oxidase in myoglobin with an O-2 reduction rate (52 s(-1)) comparable to that of a native cytochrome (cyt) cbb(3) oxidase (SO s(-1)) under identical conditions. We achieved this goal by engineering more favorable electrostatic interactions between a functional oxidase model designed in sperm whale myoglobin and its native redox partner, cyt b(5), resulting in a 400-fold electron transfer (ET) rate enhancement. Achieving high activity equivalent to that of native enzymes in a designed metalloenzyme offers deeper insight into the roles of tunable processes such as ET in oxidase activity and enzymatic function and may extend into applications such as more efficient oxygen reduction reaction catalysts for biofuel cells.

submitted time 2016-05-12 Hits1437Downloads746 Comment 0

6. chinaXiv:201605.01346 [pdf]

Genetic Incorporation of N-epsilon-Formyllysine, a New Histone Post-translational Modification

Wang, Tianyuan; Yin, Xuebin; Zhou, Qing; Li, Fahui; Wang, Jiangyun; Yu, Yang; Yin, Xuebin
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Lysine formylation is a newly discovered post-translational modification (PTM) in histones and other nuclear proteins; it has a well-recognized but poorly defined role in chromatin conformation modulation and gene expression. To date, there is no general method to site-specifically incorporate N-epsilon-formyllysine at a defined site of a protein. Here we report the highly efficient genetic incorporation of the unnatural amino acid N-epsilon-formyllysine into proteins produced in Escherichia coli and mammalian cells, by using an orthogonal N-epsilon-formyllysine tRNAsynthetase/tRNA(CUA) pair. This technique can be applied to study the role of lysine formylation in epigenetic regulation.

submitted time 2016-05-11 Hits1053Downloads590 Comment 0

7. chinaXiv:201605.01273 [pdf]

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

Lv, Xiaoxuan; Hu, Cheng; Gao, Feng; Li, Jiasong; Liu, Xiaohong; Deng, Kai; Zheng, Peng; Gong, Weimin; Wang, Jiangyun; Lv, Xiaoxuan; Hu, Cheng; Gao, Feng; Li, Jiasong; Liu, Xiaohong; Deng, Kai; Zheng, Peng; Gong, Weimin; Wang, Jiangyun; Zhou, Meng; Xia, Andong
Subjects: Biology >> Biophysics

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 (FNO(2)Phe), 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 A(0). This genetically encoded, low-reduction potential unnatural amino acid (UAA) can significantly in-Trove our ability to investigate electron transfer mechanisms in complex reductases and facilitate the design of miniature proteins that mimic their functions.

submitted time 2016-05-11 Hits1230Downloads681 Comment 0

8. chinaXiv:201605.01240 [pdf]

Fluorinated Aromatic Amino Acids Distinguish Cation-pi Interactions from Membrane Insertion

He, Tao; Gao, Jianmin; Roberts, Mary F.; Gershenson, Anne; Eyles, Stephen J.; Lee, Yan-Jiun; Liu, Wenshe R.; Wang, Jiangyun
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Cation-pi interactions, where protein aromatic residues supply pi systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-pi interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-pi interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-pi interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.

submitted time 2016-05-11 Hits846Downloads442 Comment 0

9. chinaXiv:201605.01192 [pdf]

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

Li, Fahui; Dong, Jianshu; Gong, Weimin; Li, Jiasong; Wang, Jiangyun; Li, Fahui; Dong, Jianshu; Gong, Weimin; Li, Jiasong; Wang, Jiangyun; Wang, Jiangyun; Hu, Xiaosong; Shen, Jing; Tian, Huifang
Subjects: Biology >> Biophysics

Advances in RNA research and RNA nanotechnology depend on the ability to manipulate and probe RNA with high precision through chemical approaches, both invitro 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 invitro and in mammalian cells. Subsequent click chemistry reactions facilitate the versatile labeling, functionalization, and visualization of target RNA.

submitted time 2016-05-11 Hits1222Downloads695 Comment 0

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