| Chemical biology is a blooming interdisciplinary field that uses chemical tools and chemistry principles to solve problems in cell biology,synthetic biology and so on.Genetically encoded unnatural amino acids is an outstanding representative of chemical biology,which is widely used in the field of post-translational modification of proteins,fluorescent label,protein structural analysis and others.This novel and practical method can be widely applied to E.coli,yeast,plants and animals.Currently,through introduction of Achaea ' s tyrosine tRNA/synthetase as an orthogonal pair into a cell,there are more than 80 non-natural amino acids with a variety of physical,chemical and biological properties that are encoded into the target protein with high fidelity and efficiency.Those include amino acids that have sensitive NMR signal,exhibit anomalous scattering,chelate metal ions,have novel fluorescence properties,carry various types of epigenetic modifications,have a specific photochemical properties,and have redox activity.If we compare the chemical biology as a Cartesian coordinate system,and the horizontal axis and the vertical axis is the chemistry and the life sciences disciplines respectively,the structural biology should be the cross point of this system.Structural biology is undoubtedly the most powerful weapon to explain and analyze the mechanisms in the living systems from the level of atoms,molecules and chemical bonds,especially in the field of genetic encoded unnatural amino acids.Not only the establishment of orthogonal translation system but also the screening of unnatural amino acids,is supported strongly from the structural biology.The first successful application of the tRNA/synthetase orthogonal pair to encode unnatural amino acid is Mj tRNATyr synthase from archaea jannaschii(Methanococcus jannaschii,referred Mj),which transformation is based on the structure of synthetase with the corresponding tRNA,tyrosine substrate and the ATP analogs.Methyltransferase(COMT)containing TAG stop codon encoding pyrrolysine is found from Methanosarcineae in 2002,the complex structure of pyrrolysine synthase with its substrate is also great helpful to the expanded genetic codon.The structure of aminoacyl-tRNA synthetase with a substrate or non-natural amino acids provide a reliable basis for The improvement of the library for screening amino acid aminoacyl-tRNA synthetase mutants and the catalytic efficiency of a particular synthase.Similarly,if there is significant difference in sequence and function between the wild-type protein and proteins with ant unnatural amino acids,we could explain the change of the protein conformation in the absent and present of the unusual amino acids from a biological structure perspective,and what' s more,we can explain the changes in its property and function.X-ray diffraction of the biological macromolecules,with its unique advantages,has become the mainstream tools of the field of structural biology,which has been widely used in life sciences,chemistry,medicine,pharmacy and many other areas.As a non-professional researchers in structural biology,how to make good use of this powerful technology quickly and accurately has become more and more urgent demand of us.Part of this thesis describes how to learn and master the knowledge and experimental techniques of structural biology from the perspective of a non-professional researchers.In this study,mainly under the help of the method of genetic coding of unusual amino acids,the experiments in the following include two aspects:Tyrosine phosphorylation is one of the most important of post-translational modification(PTM),which regulates enzymatic activity,protein conformation changes,and protein-protein interactions.The eukaryotic protein tyrosine kinases(PTKs)have been extensively studied in the past few decades due to their great importance in cellular signaling and diseases,and the activation mechanism of bacterial virulence is also related to the prokaryotic PTKs.Currently,most of small molecule inhibitors designed for the tyrosine kinase active center is the main method of treatment of tumors.However,with the deterioration of the disease in patients,the amino acids of tyrosine kinase that the drug targets will mutated constantly,an increasing number of patients were resistant to anticancer drugs gradually.Therefore,it is necessary to develop a more efficient and sensitive method to detect the active center of the tyrosine kinase.19F NMR spectroscopy has emerged as a powerful tool in recent years for studying the mechanism of enzyme catalysis and protein conformational changes,because it has a sensitive chemical signals in NMR,and is very sensitive to the surrounding chemical environment.Besides,there is no naturally occuring fluorine atom in protein.Researchers have developed many methods to encode fluorinated amino acids into proteins.Among them,the genetic code expansion technique has unique advantages.It can be incorporated into specific sites of the selected protein in living cells with high yield,which can be up to mg per liter of bacteria.So far,there is no report for investigating tyrosine phosphorylation with this powerful method.Here we report the highly efficient genetic incorporation of the unnatural amino acid(UAA)3,5-difluorotyrosine tyrosine,into the activation loops(a-loop)of the bacterial PTK Etk and eukaryotic PTK Src in E.Coli.These studies provide important research tool for the study of mechanism of activation of the tyrosine kinase.Such study provides new ways to screen new anticancer drugs based on the interaction between the tyrosine kinase with a substrate.We genetically incoporated the metal-chelating amino acid(S)-2-amino-3-(4-hydroxy-3-(1 H-pyrazol-1-yl)phenyl)propanoic acid(pyTyr)into green fluorescent protein(GFP).We demonstrated that Cu2+ selectively binds to pyTyr with sub-nanomolar affinity,forming a pyTyr/Cu2+ complex(termed pyTyrCu)wherein the pyrazole ring,copper ion,and phenol ring are co-planar.We showed that pyTyrCu has a redox potential of 250 mV vs.NHE,which is much lower than that of tyrosine or tryptophan.In contrast to natural amino acid or dopa,3-aminotyrosine,difluorotyorsine,which are used as electron donors in ET experiments,pyTyrCu is an electron acceptor.These unique properties should allow us to design metalloprotein sensors in ways that are previously unachievable.Using this method,we showed that PET between the GFP chromophore and pyTyrCu occurs within one nanosecond and in a distance-dependent manner,consistent with the Marcus equation.We also show that fluorescent dyes such as Cy5 and Cy7 are selectively quenched by pyTyrCu,but not by any natural amino acids.Since GFP and Cy5 are among the most important labels used for fluorescence spectroscopy,we believe that the GFP:pyTyrCu and Cy5:pyTyrCu fluorophore/quencher pairs will facilitate the design of protein PET sensors and conformational dynamics in complex biomolecules.Another route to bestow fluorescent proteins with metal binding ability is to modify the GFP chromophore through genetic code expansion.By substituting Tyr of the fluorophore in diverse fluorescent proteins(FP)with the metal-chelating UAA HqAla,we show that UAA incorporation significantly red-shifts excitation and emission spectra by more than 30 nm.We solved the X-ray structure of superfolder GFP(sfGFP)bearing HqAla in 66th position,revealing a novel 8-hydroxyquinolin-imidazolinone(HQI)chromophore which has a much expanded conjugated-system.Our results show that HqAla incorporation into the FP fluorophore bestows it with metal-chelating and metal ion sensing abilities.Among all biologically relevant metal ions,only zinc binding to HQI causes a significant increase(7.2 fold)in fluorescence.This Zn2+ selective FP sensor was then used for Zn2+ sensing in vivo.This is the first report showing that a genetically encoded metal-chelating UAA can bind transition metal ions in living cells.In the future,it would be of great interest to use HqAla to perform directed evolution in living cells in order to obtain novel metalloenzymes.Since HqAla can be synthesized through an enzymatic route in one step and in high yield,and it can bind strongly to most transition metal ions including lanthanide ions,the technological barriers which have restricted the application of the genetic encoded metal-chelating UAA have now been broken.With this method,the future is now even brighter for metalloprotein sensor engineering,metalloenzyme design,and protein NMR using paramagnetic metal ions... |
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