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Chinese Chemical Letters 2018年第10期29卷 1513-1516页

作者： Simeng Wang Yanan zhang longhua zhang Min zhang Changlin Tian School of Life Science Anhui University Hefei 230039 China National Laboratory of Physical Sciences at Microscale and School of Life Science University of Science and Technology of China Hefei 230027 China High Magnetic Field Laboratory Chinese Academy of Sciences Hefei 230031 China

Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN-remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7-HC to study the conformational change of YgaP upon SCN-binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN-. SMA is a good material to reflect the in situ conformation changes of protein than micelles.

关键词： Rhodanese YgaP Fluorescence lifetime and anisotropy SMA Conformation change

In cell measurement of fluorescence lifetime imaging microscopy revealed C-terminal conformation changes of Ferroportin upon addition of Mn^2＋

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Chinese Chemical Letters 2018年第10期29卷 1509-1512页

作者： Mengge zhang Ming Wen Ying Xiong longhua zhang Changlin Tian National Laboratory of Physical Sciences at Microscale and School of Life Sciences University of Science and Technology of China Hefei 230027 China High Magnetic Field Laboratory Chinese Academy of Sciences Hefei 230031 China

Fluorescence microscopy, as a sensitive method to detect microenvironment of molecules, is widely used in protein conformation and dynamic studies in live cells. Fluorescence lifetime imaging microscopy（FLIM）, which is independent of fluorophore concentrations, scattering and bleaching, is a suitable tool to analyze membrane proteins in a single cell. Ferroportin（FPN）, a multi-ion exporter in vertebrates, was modulated by metal ions with unknown mechanism. Herein, we fused green fluorescence protein on Cterminal of FPN（FPN-eGFP） and applied fluorescence lifetime to monitor conformation changes of FPN in a live cell. The fluorescence lifetime distribution showed a shift to shorter lifetime upon Mn^（2＋） treatment,suggesting a preference conformation of FPN in Mn^（2＋） exposure. It is also observed that the lifetime（rather than intensity） measurement was not strongly influenced by laser power. The observed fluorescence lifetime changes of FPN-eGFP upon Mn^（2＋） treatments indicated that extracellular metal ions can modulate FPN through conformation exchanges between several different states.

关键词： Fluorescence lifetime imaging microscopy In cell analysis of conformation change Ferroprotin

Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state

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Chinese Chemical Letters 2019年第5期30卷 1067-1070页

作者： Peng Zhou Pei Lv Lu Yu Sanling Liu longhua zhang Changlin Tian High Magnetic Field Laboratory Chinese Academy of Sciences and School of Life Sciences University of Science and Technology of China

Forster resonance energy transfer(FRET)is a widely used distance measurement method to illustrate protein conformational *** FRET method relies on the distance between donor and acceptor,as well as the labelling efficiency,the size and the properties of the ***,we labelled a pair of small fluorophores and calculated the energy transferred efficiency through fluorescence lifetime analysis,which can provide more reliable distance measurement than intensity *** donor fluorophore,7-hydroxycoumarin-4-yl-ethylglycine(HC),was genetically incorporated into specific sites of PYL10,obtaining complete labelling *** acceptor fluorophore,Alexa488,was labelled through the disulfide bond,whose labelling efficiency was estimated through both absorption peaks and lifetime *** lifetime and anisotropy analysis showed ABA-induced local conformation changes and dynamics of several HC incorporation sites of *** lifetime-based FRET distance measurement illustrated the conformation changes of PYL10 with or without ABA application,which is consistent with the previously reported crystal structures.

关键词： Fluorescence lifetime Anisotropy Unnatural amino acid PYL10 Distance measurement

Metabolic state oscillations in cerebral nuclei detected using two-photon fluorescence lifetime imaging microscopy

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Chinese Chemical Letters 2023年第1期34卷 300-303页

作者： Peng Zhou Jiawei Shen Jun Liang Tian Xue Yuansheng Sun longhua zhang Changlin Tian High Magnetic Field Laboratory Hefei Institutes of Physical ScienceChinese Academy of SciencesHefei 230031China The First Affiliated Hospital of USTC Division of Life Sciences and Medicineand Center for BioAnalytical ChemistryUniversity of Science and Technology of ChinaHefei 230026China ISS Inc.ChampaignIL 61822United States

The fluorescence lifetime of nicotinamide adenine dinucleotide(NADH),a key endogenous coenzyme and metabolic biomarker,can reflect the metabolic state of *** implement metabolic imaging of brain tissue at high resolution,we assembled a two-photon fluorescence lifetime imaging microscopy(FLIM)platform and verified the feasibility and stability of NADH-based two-photon FLIM in paraformaldehydefixed mouse cerebral ***,NADH based metabolic state oscillation was observed in cerebral nuclei suprachiasmatic nucleus(SCN).The free NADH fraction displayed a relatively lower level in the daytime than at the onset of night,and an ultradian oscillation at night was *** the combination of high-resolution imaging and immunostaining data,the metabolic tendency of different cell types was detected after the first two hours of the day and at ***,two-photon FLIM analysis of NADH in paraformaldehyde-fixed cerebral slices provides a high-resolution and label-free method to explore the metabolic state of deep brain regions.

关键词： NADH Fluorescence lifetime imaging microscopy Brain metabolism Metabolic oscillation High resolution

Different conformational responses of the β2-adrenergic receptor-Gs complex upon binding of the partial agonist salbutamol or the full agonist isoprenaline

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National Science Review 2021年第9期8卷 111-120页

作者： Fan Yang Shenglong Ling Yingxin Zhou Yanan zhang Pei Lv Sanling Liu Wei Fang Wenjing Sun Liaoyuan A.Hu longhua zhang Pan Shi Changlin Tian Hefei National Laboratory of Physical Sciences at Microscale and School of Life Sciences University of Science and Technology of China Amgen Asia R&D Center Amgen Research Shanghai 201210 China3High Magnetic Field Laboratory Chinese Academy of Sciences

G protein-coupled receptors(GPCRs) are responsible for most cytoplasmic signaling in response to extracellular ligands with different efficacy profiles. Various spectroscopic techniques have identified that agonists exhibiting varying efficacies can selectively stabilize a specific conformation of the ***, the structural basis for activation of the GPCR-G protein complex by ligands with different efficacies is incompletely understood. To better understand the structural basis underlying the mechanisms by which ligands with varying efficacies differentially regulate the conformations of receptors and G proteins, we determined the structures of β2AR-Gαsβγ bound with partial agonist salbutamol or bound with full agonist isoprenaline using single-particle cryo-electron microscopy at resolutions of 3.26 ?A and3.80 ?A, respectively. Structural comparisons between the β2AR-Gs-salbutamol and β2AR-Gs-isoprenaline complexes demonstrated that the decreased binding affinity and efficacy of salbutamol compared with those of isoprenaline might be attributed to weakened hydrogen bonding interactions, attenuated hydrophobic interactions in the orthosteric binding pocket and different conformational changes in the rotamer toggle switch in TM6. Moreover, the observed stronger interactions between the intracellular loop 2 or 3(ICL2 or ICL3) of β2AR and Gαswith binding of salbutamol versus isoprenaline might decrease phosphorylation in the salbutamol-activated β2AR-Gs complex. From the observed structural differences between these complexes of β2AR, a mechanism of β2AR activation by partial and full agonists is proposed to provide structural insights into β2AR desensitization.

关键词： cryo-EM structure G protein-coupled receptor (GPCR) partial and full agonists conformational change desensitization

Protein-protein interaction analysis in crude bacterial lysates using combinational method of ^19F site-specific incorporation and ^19F NMR

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Protein & Cell 2017年第2期8卷 149-154页

作者： Dong Li Yanan zhang Yao He Chengwei zhang Jiefei Wang Ying Xiong longhua zhang Yangzhong Liu Pan Shi Changlin Tian Hefei National Laboratory for Physical Science at the Microscale School of Life Science University of Science and Technology of China Hefei 230026 Anhui China School of Chemistry University of Science and Technology of China Hefei 230026 Anhui China

Protein-protein interactions （PPI） are essential for a variety of cellular functions. Many PPI analyses were conducted in vitro, using purified proteins. In this report, the unnatural amino acid tfmF was site-specifically incorporated into several different sites of two Phox-Beml （PB1） domains from two mitogen activated protein kinases （MEKK3 and/ or MEK5） in the E. coil cells.

关键词： bacterial interaction Protein-protein

Mutation of the critical pH-gating residues histidine 231 to glutamate increase open probability of outer membrane protein G in planar lipid bilayer

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Protein & Cell 2013年第11期4卷 803-806页

作者： Mu Yu Peibei Sun Yao He Liang Xiao Demeng Sun longhua zhang Changlin Tian High Magnetic Field Laboratory Chinese Academic of SciencesHefei 230031China Hefei National Laboratory for Physical Science at Microscale and School of Life Science University of Science and Technology of ChinaHefei 230027China

Dear Editor,Outer-membrane protein G(OmpG)is a nonspecificβ-barrel porin in the outer membrane of Escherichia coli(***),allowing the passage of ions and molecules up to 900 Da(Fajardo et al.,1998).It comprises of 280 amino acids that form 14-strandedβ-sheets with seven long loops(L1-L7)on the extra-cellular side and six short turns on the periplasmic side(Subbarao and van den Berg,2006;Yildiz et al.,2006;Liang and Tamm,2007).Despite that the OmpG gene exists in the genome of several *** strains(Nikaido,1999),expres-sion of OmpG was only observed in *** mutants lacking OmpF and LamB(Fajardo et al.,1998)to enable the diffu-sion of maltodextrins across the bacte-rial outer *** interestingly,unlike usual trimeric channel-forming porins,OmpG exhibits fascinating characteristics of a functional monomer in physiological and structural studies(Conlan and Bayley,2003;Mari et al.,2010).

关键词： outer planar residues

Distance measurement between two flexible sites in proteins in high viscosity medium at physiological temperature using continuous wave EPR

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Protein & Cell 2014年第5期5卷 334-337页

作者： Lu Yu Wei Wang Shenglong Ling Yao He Liang Xiao Kaiqi Wu longhua zhang Changlin Tian Hefei National Laboratory for Physical Science at the Microscale School of Life Science University of Science and Technology of ChinaHefei 230026China High Magnetic Field Laboratory Chinese Academy of SciencesHefei 230031China

Dear Editor, Accurate measurement of distances within and between biological macromolecules is important for structure-function studies, for investigating conformational changes and dynamics, and for improving restraints for molecular dynamics and modeling programs （Hellmich and Glaubitz, 2009）. Several methods have been developed to measure the distances between atoms, residues, and domains in single proteins and larger complexes. Fluorescence reso- nance energy transfer （FRET） is currently the most fre- quently used method to derive distances in cells or protein complexes, but the bulky size of the fluorescence probes and the strict requirement for frequency profile overlap between the acceptor and donor chromophores are limita- tions in many cases （Prevo and Peterman, 2014）.

关键词： Distance high viscosity EPR

Climate change:Impact on the Arctic,Antarctic and Tibetan Plateau

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Advances in Polar Science 2011年第2期22卷 67-73页

作者： LU longhua BIAN LinGen zhang ZhengQiu Chinese Academy of Meteorological Sciences Beijing 100081 China

The Arctic, Antarctic and Tibetan Plateau are very sensitive to global climate change. Hence, it is urgent that we improve our understanding ot＇ how they respond to climate change, and how those responses in turn affect both regional and global climate. Against a background of current global warming, the three poles display climate diversities temporarily and spatially, which to different degrees affect the weather and climate over China. Enhanced monitoring of climate change in these three areas, as well as connected work on the responses and feedbacks of the three regions to climate change, will provide necessary support for adaptation and the sustainable development of the Chinese economy.

关键词： Three poles Tibetan Plateau climate change global change scientific research climate change adaptation

Nano-size uni-lamellar lipodisq improved in situ auto-phosphorylation analysis of E. coli tyrosine kinase using 19F nuclear magnetic resonance

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Protein & Cell 2015年第3期6卷 229-233页

作者： Dong Li(1) Juan Li(1) Yonglong Zhuang(3) longhua zhang(1) Ying Xiong(1) Pan Shi (2) Changlin Tian (1)(2) 1.Hefei National Laboratory for Physical Science at the Microscale & School of Life Science University of Science and Technology of China Hefei 230026 China 3.Department of Chemistry Anhui University Hefei 230027 China 2.High Magnetic Field Laboratory Chinese Academy of Sciences Hefei 230031 China

Dear Editor, Among many of biophysical methods, 19F NMR spectros- copy has emerged as a powerful tool for characterizing protein structure, dynamics and function properties due to high intrinsic sensitivities of fluorine, 100% natural abundance of the NMR-active spin, the absence of any natural background in cells, and exquisite sensitivities of 19F chemical shift to environment （Shi et al., 2011; Shi et al.,

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