In the English language classroom, authenticity is a basic discoursal feature of teachers' instructional discourse. Without authenticity, there will be a mismatch between what students learn in the classroom and what...In the English language classroom, authenticity is a basic discoursal feature of teachers' instructional discourse. Without authenticity, there will be a mismatch between what students learn in the classroom and what they use in real life. However, the notion of authenticity varies with the change between the natural mode and pedagogic mode in the classroom. Therefore, based on this assumption, retrieving and analyzing data from the corpus and classroom videos, the present paper identifies two categories of problems in Chinese teachers' instructional discourse. Sample extracts are discussed and predictions are made on how teachers' failure to produce authentic discourse may result in ineffective teaching and learning.展开更多
Polyketides have been widely used clinically due to their significant biological activities, but the needed structural and functional diversity cannot be achieved by common chemical synthetic methods. The tool of comb...Polyketides have been widely used clinically due to their significant biological activities, but the needed structural and functional diversity cannot be achieved by common chemical synthetic methods. The tool of combinatorial biosynthesis provides the possibility to produce "unnatural" natural drugs, which has achieved initial success. This paper provides an overview for the strategies of combinatorial biosynthesis in producing the structural and functional diversity of polyketides, including the redesign of metabolic flow, polyketide synthase(PKS) engineering, and PKS post-translational modification. Although encouraging progress has been made in the last decade, challenges still exist regarding the rational combinatorial biosynthesis of polyketides. In this review, the perspectives of polyketide combinatorial biosynthesis are also discussed.展开更多
The coupled-channels optical method for positron scattering has been applied to investigate resonance states with unnatural parities in a positron-excited hydrogen system. The positronium formation channels and contin...The coupled-channels optical method for positron scattering has been applied to investigate resonance states with unnatural parities in a positron-excited hydrogen system. The positronium formation channels and continuum channel are included via a complex equivalent local potential. Resonance states with angular momenta L =- 1 to L = 2 and parities (-1)L+1 are calculated. Resonance energies and widths are reported and compared with other theoretical calculations. We found that the opening positronium formation channels play an important role in forming nondipole Feshbach resonances.展开更多
Expectation values of single electron and interelectronic geometric quantities such as <γ>,<γ_(12)> ,<γ_(<)> , <γ_(>)>,<cosθ_(12)> and <θ_(12)> are calculated for doubly...Expectation values of single electron and interelectronic geometric quantities such as <γ>,<γ_(12)> ,<γ_(<)> , <γ_(>)>,<cosθ_(12)> and <θ_(12)> are calculated for doubly excited 2pnp ^(1)P^(e)(3 ≤ n ≤ 5), 2pnp ^(3)P^(e)(2 ≤ n ≤ 5)and 2pnd ^(1,3)D°(3 ≤ n ≤ 5) states of helium using Hylleraas-B-spline basis set. The energy levels converge to at least 10 significant digits in our calculations.The extrapolated values of geometric quantities except for <θ_(12)> reach 10 significant digits as well;<θ_(12)> reaches at least 7 significant digits using a multipole expansion approach. Our results provide a precise reference for future research.展开更多
Dear Editor, In 2011, Son et al. (2011) reported that the forced expression of selected transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). The authors use...Dear Editor, In 2011, Son et al. (2011) reported that the forced expression of selected transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). The authors used three factors (Ascll, Brn2, and Mytll) to convert fibroblasts into neuronal-like ceils. After confirming that the cells had neuronal morphology, but with absence of motor neuron markers, eight candidate transcription factors were added, which participate in various stages of motor neuron specification. As expected, a significant number of motor cells emerged with known characteristics of cultured embryonic motor neurons.展开更多
Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a la...Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a lack of direct and simple methods.To address this challenge,we designed the LADA(labeling-activation-desulfurization-addition)strategy:selective labeling and activation of cysteine residues,the photocatalytic desulfurization and the subsequent radical addition to alkenes.Although composed of two steps,it is one-pot synthesis and has advantages such as high functional group tolerance,biocompatible reaction condition,and retained stereochemistry.This highly efficient strategy was successfully applied in the direct synthesis of unnatural amino acids and modifications of peptides with more than 50 examples.展开更多
Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansio...Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.展开更多
Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified s...Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified synthesis of UAAs remains a great challenge.At the same time,vast known peptide modifications are based on carbon-heteroatom bonds.There are no general methods for peptide modifications via the construction of C–C bonds.To address this challenge,herein we propose the LADA strategy,which is composed of two steps:the selective labeling and activation of cysteine residues,the desulfurization to generate carbon-centered radical and the radical addition to alkenes to build C–C bond.This one-pot protocol has obvious advantages such as good functional group tolerance,biocompatible reaction conditions,and retained stereochemistry.This strategy was successfully utilized for the synthesis of unnatural amino acids and direct modifications of peptides.展开更多
China first in-situ lunar dust experiment is performed by a lunar dust detector in Chang’E-3 mission. The existed dust(less than 20 μm in diameter) properties, such as levitation, transportation and adhesion, are cr...China first in-situ lunar dust experiment is performed by a lunar dust detector in Chang’E-3 mission. The existed dust(less than 20 μm in diameter) properties, such as levitation, transportation and adhesion, are critical constraints for future lunar exploration program and even manned lunar exploration. Based on the problems discussed above, the in-situ lunar dust detector is originally designed to characterize dust deposition properties induced by lander landing as a function of environmental temperature, solar incident angle and orbit short circuit current on the northern Mare Imbrium, aiming to study lunar dust deposition properties induced by lander landing in depth. This paper begins with a brief of introduction of Chang’E-3 lunar dust detector design,followed by a series of experimental analysis of this instrument under different influencing factors, and concludes with lunar dust mass density deposition amount observed on the first lunar day is about 0.83 mg/cm^2, which is less than that observed in Apollo 11 mission because the landing site of Chang’E-3 has the youngest mare basalts comparing with previous Apollo and lunar landing sites. The young geologic environment is less weathered and thus it has thinner layer of lunar dust than Apollo missions’;hence, the amount of kicked-up lunar dust in Chang’E-3 mission is less than that in Apollo 11 mission.展开更多
Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster...Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.展开更多
Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency...Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.展开更多
Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered a...Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.展开更多
Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthes...Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthesis of diverse natural products. Accordingly the development of cost-effective approaches for the preparation of unnatural a-amino acids has received increasing attentions. Among all the available methods for this purpose, direct C–H functionalization of simple amino acids represents one of the most attractive approaches because it exhibits good atom-economy and step-efficiency. In particular, selective functionalization of either the primary or secondary C(sp^3)–H bonds in the amino acids has been explored to make versatile C–C, C–N, C–O, C–B and C–F bonds to modify the side chain of amino acids and even peptides. The present review surveys the recent advances of synthesis of chiral unnatural a-amino acids and peptides through palladium-catalyzed functionalization of un-activated C(sp^3)–H bonds.展开更多
Forster resonance energy transfer(FRET)is a widely used distance measurement method to illustrate protein conformational dynamics.The FRET method relies on the distance between donor and acceptor,as well as the labell...Forster resonance energy transfer(FRET)is a widely used distance measurement method to illustrate protein conformational dynamics.The FRET method relies on the distance between donor and acceptor,as well as the labelling efficiency,the size and the properties of the fluorophores.Here,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 attenuation.The donor fluorophore,7-hydroxycoumarin-4-yl-ethylglycine(HC),was genetically incorporated into specific sites of PYL10,obtaining complete labelling efficiency.The acceptor fluorophore,Alexa488,was labelled through the disulfide bond,whose labelling efficiency was estimated through both absorption peaks and lifetime populations.Fluorescence lifetime and anisotropy analysis showed ABA-induced local conformation changes and dynamics of several HC incorporation sites of PYL10.The 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.展开更多
Post translational modifications (PTMs) of lysine play a crucial role on modulating the activity and stability of essential proteins in eukaryotic cells including the tumor
Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-cod...Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.展开更多
文摘In the English language classroom, authenticity is a basic discoursal feature of teachers' instructional discourse. Without authenticity, there will be a mismatch between what students learn in the classroom and what they use in real life. However, the notion of authenticity varies with the change between the natural mode and pedagogic mode in the classroom. Therefore, based on this assumption, retrieving and analyzing data from the corpus and classroom videos, the present paper identifies two categories of problems in Chinese teachers' instructional discourse. Sample extracts are discussed and predictions are made on how teachers' failure to produce authentic discourse may result in ineffective teaching and learning.
基金supported by the Major Research Plan of Tianjin(No.16YFXTSF00460)
文摘Polyketides have been widely used clinically due to their significant biological activities, but the needed structural and functional diversity cannot be achieved by common chemical synthetic methods. The tool of combinatorial biosynthesis provides the possibility to produce "unnatural" natural drugs, which has achieved initial success. This paper provides an overview for the strategies of combinatorial biosynthesis in producing the structural and functional diversity of polyketides, including the redesign of metabolic flow, polyketide synthase(PKS) engineering, and PKS post-translational modification. Although encouraging progress has been made in the last decade, challenges still exist regarding the rational combinatorial biosynthesis of polyketides. In this review, the perspectives of polyketide combinatorial biosynthesis are also discussed.
基金Project supported by the National Natural Science Foundation of China(Grant No.10674055)
文摘The coupled-channels optical method for positron scattering has been applied to investigate resonance states with unnatural parities in a positron-excited hydrogen system. The positronium formation channels and continuum channel are included via a complex equivalent local potential. Resonance states with angular momenta L =- 1 to L = 2 and parities (-1)L+1 are calculated. Resonance energies and widths are reported and compared with other theoretical calculations. We found that the opening positronium formation channels play an important role in forming nondipole Feshbach resonances.
基金Project supported by the National Natural Science Foundation of China (Grant No. 12074295)。
文摘Expectation values of single electron and interelectronic geometric quantities such as <γ>,<γ_(12)> ,<γ_(<)> , <γ_(>)>,<cosθ_(12)> and <θ_(12)> are calculated for doubly excited 2pnp ^(1)P^(e)(3 ≤ n ≤ 5), 2pnp ^(3)P^(e)(2 ≤ n ≤ 5)and 2pnd ^(1,3)D°(3 ≤ n ≤ 5) states of helium using Hylleraas-B-spline basis set. The energy levels converge to at least 10 significant digits in our calculations.The extrapolated values of geometric quantities except for <θ_(12)> reach 10 significant digits as well;<θ_(12)> reaches at least 7 significant digits using a multipole expansion approach. Our results provide a precise reference for future research.
基金2013 Six Peak Talents in Jiangsu Province,Project No.WSN-022333 Key Talents of Science and Technology in Jiangsu Province,Project No.BRA2012094+1 种基金Scientific Project of Traditional Chinese Medicine of Jiangsu Province,Project No.LZ131952011 Project of Scientific Technology and Social Development in Yangzhou,Project No.YZ2011084
文摘Dear Editor, In 2011, Son et al. (2011) reported that the forced expression of selected transcription factors is sufficient to convert mouse and human fibroblasts into induced motor neurons (iMNs). The authors used three factors (Ascll, Brn2, and Mytll) to convert fibroblasts into neuronal-like ceils. After confirming that the cells had neuronal morphology, but with absence of motor neuron markers, eight candidate transcription factors were added, which participate in various stages of motor neuron specification. As expected, a significant number of motor cells emerged with known characteristics of cultured embryonic motor neurons.
文摘Unnatural amino acids(UAAs)have broad applications in pharmaceutical sciences and biological studies.Current synthetic methods for UAAs mainly rely on asymmetric catalysis and often require several steps.There is a lack of direct and simple methods.To address this challenge,we designed the LADA(labeling-activation-desulfurization-addition)strategy:selective labeling and activation of cysteine residues,the photocatalytic desulfurization and the subsequent radical addition to alkenes.Although composed of two steps,it is one-pot synthesis and has advantages such as high functional group tolerance,biocompatible reaction condition,and retained stereochemistry.This highly efficient strategy was successfully applied in the direct synthesis of unnatural amino acids and modifications of peptides with more than 50 examples.
基金This work was supported by the National Key R&D Program of China(Nos.2019YFA0904200 and 2019YFA0906100)the National Natural Science Foundation of China(No.32171464)Shenzhen Science and Technology Innovation Program(JCYJ20180504165501371).
文摘Unnatural amino acids(UAAs)have gained significant attention in protein engineering and drug development owing to their ability to introduce new chemical functionalities to proteins.In eukaryotes,genetic code expansion(GCE)enables the incorporation of UAAs and facilitates posttranscriptional modification(PTM),which is not feasible in prokaryotic systems.GCE is also a powerful tool for cell or animal imaging,the monitoring of protein interactions in target cells,drug development,and switch regulation.Therefore,there is keen interest in utilizing GCE in eukaryotic systems.This review provides an overview of the application of GCE in eukaryotic systems and discusses current challenges that need to be addressed.
基金supported by the Shanghai Jiao Tong University(WF220417003 to Z.S.)。
文摘Unnatural amino acids(UAAs)are important building blocks in organic synthesis and drug discovery.They are also frequently integrated into peptides or proteins for biological studies.However,the direct and simplified synthesis of UAAs remains a great challenge.At the same time,vast known peptide modifications are based on carbon-heteroatom bonds.There are no general methods for peptide modifications via the construction of C–C bonds.To address this challenge,herein we propose the LADA strategy,which is composed of two steps:the selective labeling and activation of cysteine residues,the desulfurization to generate carbon-centered radical and the radical addition to alkenes to build C–C bond.This one-pot protocol has obvious advantages such as good functional group tolerance,biocompatible reaction conditions,and retained stereochemistry.This strategy was successfully utilized for the synthesis of unnatural amino acids and direct modifications of peptides.
基金supported by the Beijing Institute of Spacecraft System Engineeringthe National Natural Science Foundation of China(Grant No.11605080)the State Key Laboratory of Environmental Geochemistry for providing the simulant lunar dust
文摘China first in-situ lunar dust experiment is performed by a lunar dust detector in Chang’E-3 mission. The existed dust(less than 20 μm in diameter) properties, such as levitation, transportation and adhesion, are critical constraints for future lunar exploration program and even manned lunar exploration. Based on the problems discussed above, the in-situ lunar dust detector is originally designed to characterize dust deposition properties induced by lander landing as a function of environmental temperature, solar incident angle and orbit short circuit current on the northern Mare Imbrium, aiming to study lunar dust deposition properties induced by lander landing in depth. This paper begins with a brief of introduction of Chang’E-3 lunar dust detector design,followed by a series of experimental analysis of this instrument under different influencing factors, and concludes with lunar dust mass density deposition amount observed on the first lunar day is about 0.83 mg/cm^2, which is less than that observed in Apollo 11 mission because the landing site of Chang’E-3 has the youngest mare basalts comparing with previous Apollo and lunar landing sites. The young geologic environment is less weathered and thus it has thinner layer of lunar dust than Apollo missions’;hence, the amount of kicked-up lunar dust in Chang’E-3 mission is less than that in Apollo 11 mission.
文摘Cell-free synthetic biology emerges as a powerful and flexible enabling technology that can engineer biological parts and systems for life science applications without using living cells.It provides simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.This review focuses on recent developments of cell-free synthetic biology on biological engineering fields at molecular and cellular levels,including protein engineering,metabolic engineering,and artificial cell engineering.In cell-free protein engineering,the direct control of reaction conditions in cell-free system allows for easy synthesis of complex proteins,toxic proteins,membrane proteins,and novel proteins with unnatural amino acids.Cell-free systems offer the ability to design metabolic pathways towards the production of desired products.Buildup of artificial cells based on cell-free systems will improve our understanding of life and use them for environmental and biomedical applications.
基金supported by the National Key R&D Program of China(2018YFA0901700)National Natural Science Foundation of China(21878173)+2 种基金Beijing Natural Science Foundation(2192023)a grant from the Institute Guo Qiang,Tsinghua University(2019GQG1016)Laboratory Innovation Fund of Tsinghua University.
文摘Site-specific incorporation of unnatural amino acids(UNAAs)into proteins using an orthogonal translation system(OTS)has expanded the scope of protein-coding chemistry.The key factor affecting UNAA embedding efficiency is the orthogonality of the OTS.Compared to traditional cell systems,cell-free systems are more convenient to control the reaction process and improve the utilization rate of UNAA.In this study,a linear DNA template-based cell-free unnatural protein synthesis system for rapid high-throughput screening and evolution was proposed.A total of 14 cell extracts were selected for screening out cell extract with high expression level.The result showed that EcAR7ΔAΔSer cell extract was optimal for the cell-free system.In addition,the screening results of four UNAAs,p-propargyloxy-l-phenylalanine(pPaF),p-azyl-phenylalanine(pAzF),p-acetyl-l-phenylalanine(pAcF),and p-benzoyl-l-phenylalanine(pBpF),showed that o-aaRS and o-tRNA of pPaF had good orthogonality.A new pair of corresponding o-aaRS and o-tRNA for pBpF was screened out.These results proved that this method could speed up the screening of optimal OTS components for UNAAs with versatile functions.
基金We acknowledge support from The Camille and Henry Dreyfus Teacher Scholar Program,The Welch Foundation under Grant F-1753,and the Air Force Office of Scientific Research under Award No.FA9550-14-1-0089.
文摘Metabolic engineering offers an exquisite capacity to produce new molecules in a renewable manner.However,most industrial applications have focused on only a small subset of elements from the periodic table,centered around carbon biochemistry.This review aims to illustrate the expanse of chemical elements that can currently(and potentially)be integrated into useful products using cellular systems.Specifically,we describe recent advances in expanding the cellular scope to include the halogens,selenium and the metalloids,and a variety of metal incorporations.These examples range from small molecules,heteroatom-linked uncommon elements,and natural products to biomining and nanotechnology applications.Collectively,this review covers the promise of an expanded range of elemental incorporations and the future impacts it may have on biotechnology.
文摘Unnatural a-amino acids have been extensively used in the modern drug discovery and protein engineering studies. They have also found applications in the development of chiral molecular catalysts and the total synthesis of diverse natural products. Accordingly the development of cost-effective approaches for the preparation of unnatural a-amino acids has received increasing attentions. Among all the available methods for this purpose, direct C–H functionalization of simple amino acids represents one of the most attractive approaches because it exhibits good atom-economy and step-efficiency. In particular, selective functionalization of either the primary or secondary C(sp^3)–H bonds in the amino acids has been explored to make versatile C–C, C–N, C–O, C–B and C–F bonds to modify the side chain of amino acids and even peptides. The present review surveys the recent advances of synthesis of chiral unnatural a-amino acids and peptides through palladium-catalyzed functionalization of un-activated C(sp^3)–H bonds.
基金supported by National Key R&D Program of China(Nos.2017YFA0505300,2016YFA0400900)the Instrument Developing Project of the Chinese Academy of Sciences(No.YZ201564)the National Natural Science Foundation of China(Nos.U1832181,31670776,31500611)
文摘Forster resonance energy transfer(FRET)is a widely used distance measurement method to illustrate protein conformational dynamics.The FRET method relies on the distance between donor and acceptor,as well as the labelling efficiency,the size and the properties of the fluorophores.Here,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 attenuation.The donor fluorophore,7-hydroxycoumarin-4-yl-ethylglycine(HC),was genetically incorporated into specific sites of PYL10,obtaining complete labelling efficiency.The acceptor fluorophore,Alexa488,was labelled through the disulfide bond,whose labelling efficiency was estimated through both absorption peaks and lifetime populations.Fluorescence lifetime and anisotropy analysis showed ABA-induced local conformation changes and dynamics of several HC incorporation sites of PYL10.The 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.
基金The US National Institute of Health R01 GM062159 and the Skaggs Institute for Chemical Biology
文摘Post translational modifications (PTMs) of lysine play a crucial role on modulating the activity and stability of essential proteins in eukaryotic cells including the tumor
基金the National Key R&D Program of China(Grant No.2018YFA0901700)the National Natural Science Foundation of China(Grant No.21878173)the Beijing Natural Science Foundation(Grant No.2192023).
文摘Amber suppression has been widely used to incorporate unnatural amino acids(UNAAs)with unique structures or functional side-chain groups into specific sites of the target protein,which expands the scope of protein-coding chemistry.However,this traditional strategy does not allow multiple-site incorporation of different UNAAs into a single protein,which limits the development of unnatural proteins.To address this challenge,the suppression method using multiple termination codons(TAG,TAA or TGA)was proposed,and cell-free unnatural protein synthesis(CFUPS)system was employed.By the analysis of incorporating 3 different UNAAs(p-propargyloxy-L-phenylalanine,p-azyl-phenylalanine and L-4-Iodophenylalanine)and mass spectrometry,the simultaneous usage of the codons TAG and TAA were suggested for better multiple-site UNAA incorporation.The CFUPS conditions were further optimized for better UNAA incorporation efficiency,including the orthogonal translation system(OTS)components,magnesium ions,and the redox environment.This study established a CFUPS approach based on multiple termination codon suppression to achieve efficient and precise incorporation of different types of UNAAs,thereby synthesizing unnatural proteins with novel physicochemical functions.