《蛋白质及其标记》PPT课件.ppt

Advanced Bioanalytical Chemistry Fall, 2016 朱 志 卢嘉锡 542 第六节课 生物分子的化学标记和探针技术 2016年11月3日,生物分子的化学标记和探针技术,Some of the common bioconjugate designs often used for life science applications include: (A) streptavidinenzyme conjugate (B) an immobilized affinity ligand on a particle (C) an oligo molecular beacon probe containing two fluorescent labels or a fluor and a quencher at each end (D) fluorescently labeled streptavidin (E) an affinity ligand attached to a surface (F) a biotinylated enzyme (G) an antibodyenzyme conjugate (H) a fluorescently labeled antibody (I) a biotinylated antibody (J) a biotinylated oligo probe (K) an antibodydrug conjugate (L) a gadolinium chelate-modified dendrimer containing folate molecules for targeting.,蛋白质和核酸的荧光标记,Localization of Molecules with Fluorescence Microscopy,Image from Molecular Probes (Invitrogen): ,Fluorescence Applications in Biology,Fluorescence Techniques Can Provide the Following Information: Simple fluorophores: - Localization of biomolecules - Assessment of binding - Calculation of diffusion coefficients More complex systems: - Reporting of gene activation - Detection of physiological changes - Measurement of distances and conformational changes,Examples of Fluorescent Molecules,Fluorescein (Fluorescent) lex = 494, lem = 518,Phenolphthalein (Non-fluorescent),Pyrene lex = 345, lem = 378,Coumarin 343 lex = 454, lem = 495,BODIPY 493 lex = 500, lem = 506,TAMRA lex = 555, lem = 580,Texas Red lex = 595, lem = 615,7-Hydroxycoumarin lex = 385, lem = 445,Cy5 lex = 643, lem = 667,Fluorescence of native proteins residues: Tryptophan: lex = 290, lem = 330-350, depending on environment DNA and RNA generally exhibit no fluorescence.,Partial energy diagram for a photoluminescent system.,荧光的产生,Spectral observables for fluorescence sensing. From left to right: Intensity, intensity ratio, anisotropy, time-domain lifetime, and phase-modulation lifetime.,蛋白质的选择性标记,New Functionality,Chromophores,Affinity Labels,Spin Labels,Catalysts,Crosslinkers,Polymers,Enzymes,Nanocrystals,MRI Contrast Agents,Material Surfaces,Chemical Reaction,Radiolabels,Common Examples of Protein Bioconjugates,氨基的反应（赖氨酸侧链与N末端氨基）,理想反应条件：pH 89,质子化率的计算： lg(质子受体/质子供体)=pH-pK Lys -+NH3 pK=10.5 求- +NH3 在pH9.5及pH=11时的质子化%。,解: - +NH3 = -NH2 + H + 根据公式：pH=pK+lg(质子受体/质子供体) 9.5=10.5 +lg(-NH2 /- + NH3 ) lg(- NH2 /-+NH3 )=-1 -NH2 /-+NH3 =1/10 质子化%=10/(10+1)=91% PH=11时，同理可得，质子化%24,典型的NHS酯类荧光标记试剂,典型的NHS酯类荧光标记试剂,氨基的反应（赖氨酸侧链与N末端氨基）,异硫氰酸酯比羟基琥柏酰亚胺酯在水溶液中更稳定 理想反应条件：pH 99.5,异硫氰酸酯的反应,Schiff碱不稳定，需要进一步还原,醛基的反应（Schiff碱）,氨基的反应（赖氨酸侧链与N末端氨基）,巯基的反应（半胱氨酸侧链） 与卤乙酰胺反应,中性条件 在氨基的存在下也可选择性与巯基反应 反应速度：碘乙酰衍生物是溴乙酰衍生物的2倍、氯溴乙酰衍生物的100倍 硫醚键产物稳定,巯基的反应（半胱氨酸侧链） 与顺丁烯二酰亚胺（maleimide）反应,比碘乙酰胺选择性更高 中性条件pH 7, pH 高于8水解成不反应的顺丁烯二酸 在氨基的存在下也可选择性与巯基反应,氨基与羧基的反应 Carbodiimides (N=C=N) 碳酰二亚胺法,Mediate the formation of amide linkages between a carboxylate and an amine, or phosphoramidate linkages between a phosphate and an amine. Most popular - two protein molecules, a peptide and a protein, oligonucleotides and proteins, etc. 水溶/非水溶 - 生物大分子/多肽、有机合成 EDC, EDC plus Sulfo-NHS, CMC, DCC, DIC,EDC (1-(3-二甲氨基丙基)-3-乙基碳二亚胺),水溶性好，多余试剂、副产物易去除 水中不稳定，-20冻干保存。用时放至室温后速取速溶速用。,Mechanism,EDC plus Sulfo-NHS,Water-soluble, long-lived, 相对水解缓慢 The advantage of adding Sulfo-NHS - 稳定中间产物防其水解，提高产率,DCC(二环己基碳二亚胺常溶有机用于有机合成),Problems: 副产物N,N-二环己基脲不溶于水,Bioconjugate Techniques, Third Edition 3rd Edition by Greg T. Hermanson (Author),Biological Labeling with Green Fluorescent Protein (GFP),-GFP (or the newer EGFP) is a 27 kD (236 residue) protein isolated from the jellyfish Aequorea victoria. -The structure consists of an 11-stranded b-barrel surrounding a single central strand. -After the protein folds, three residues spontaneously cyclize in an unprecedented fashion. -A subsequent oxidation (non-enzymatic) results in the formation of a strong fluorescent chromophore. -Although the wt protein exhibits green fluorescence, blue, cyan, and red mutants have now been identified. -The chromophore does not fluoresce when removed from the protein core.,Protein folding Cyclization Oxidation,Roger Tsien UCSD,双砷染料-四半胱氨酸体系,与连载重组蛋白质末端的六肽标签CCXXCC序列（TC-tag）特异性标记,Roger Tsien UCSD,双砷染料-四半胱氨酸体系的荧光强度是双砷染料的 50000 倍。 跟荧光蛋白相比，四半胱氨酸标签序列体积小，不会影响被标记蛋白质 或细胞的正常生理功能，另外染料量子产率高、稳定性强、标记速度快,ReAsH (a phenoxaine derivative),CHOxAsH (a dihydroxyxanthone derivative),Tsien et al. Science 1998, 281, 269-271. and JACS 2002, 124, 6063-6076.,双砷染料-四半胱氨酸体系,双砷染料-四半胱氨酸重组噬菌体的构建及 应用,Angew. Chem. Int. Ed., 2011, 50, 5873-5877,其他特殊标记方法： NTA荧光团缀合物探针,由两部分组成，一部分为荧光基团，另一部分为螯合物， 由金属离子与含有三醋酸根的叔胺类化合物组成（NTA） 六组氨酸(His6 ) 识别结合 Ni2+：次氮基三乙酸（Ni2+:NTA) 蛋白质C端、N端、或肽链中间 比双砷染料-四半胱氨酸体系通用,The term click chemistry, originally brought up by K. Barry Sharpless at The Scripps Research Institute in 2001.,K. Barry Sharpless and his co-workers have discovered and developed many widely used catalytic oxidation processes, the Sharpless reactions for asymmetric epoxidation, dihydroxylation, and aminohydroxylation of olefins （烯烃的不对称环氧化、双羟化和羟氨化反应）.,The Nobel Prize, 2001,点击化学,Azide/Alkyne Cycloaddition,L. Pauling. Proc. Natl. Acad. Sci. USA 1933, 19, 860-867; Huisgen, R. Angew. Chem. Int. Ed. 1963, 2, 633-696 Sharpless, K.B. et al. Angew. Chem. Int. Ed 2002, 41, 2596-2599; Meldal,M.J. et al. J. Org. Chem. 2002, 67, 3057-3064,1933- Dipolar nature of azide first recognized by Linus Pauling,1960- Mechanism of 1,3-dipolar cycloaddition of azides and alkynes pioneered by Rolf Huisgen,2001- Copper catalyzed 1,3-Dipolar cycloaddition by Sharpless/Meldal,Strain-Promoted 3 + 2 Azide-Alkyne Cycloaddition,Cu()催化的端基炔和叠氮化物Huisgen 1，3-偶极环加成反应(CuAAC)，CuAAC虽有很高的区域选择性并且反应迅速，但是在终产物中会存在微量的不希望有的铜盐。由于铜的细胞毒性和伴随生理调节(attendant bioregulation)，铜催化的反应还未能直接应用于活体细胞中,Agard N J, Prescher J A, Bertozzi C R,J. Am. Chem. Soc. ,2004, 126,15046,环辛炔衍生物提高水溶性与反应速率,In vivo imaging,Strain-Promoted 3 + 2 Azide-Alkyne Cycloaddition,无需铜离子的点击反应,Staudinger-Bertozzi反应,Saxon, E. & Bertozzi, C.R. Science 287, 20072010 (2000).,Staudinger ligation reaction scheme (azide-phosphine conjugation). Phosphine-activated proteins or labeling reagents react with azide-labeled target molecules to form aza-ylide intermediates that quickly rearrange in aqueous conditions to form stable amide bonds between reactant molecules.,Chemoselectivity: phosphines and azides do not occur in cells, they react only with each other in biological samples, resulting in minimal background and few artifacts,Compatibility with the design and use of bioorthogonal derivatives. Azide group is extremely small, amino acids, sugars and other building blocks for metabolism can be synthesized to contain azide groups and then supplied to cells as substitutes of their natural counterparts. The selectable azide tag can be incorporated into molecules of interest by the cellular metabolic machinery, allowing the underlying metabolic activity to be detected and measured via phosphine-activated reagents. Phosphine activation of proteins is easily accomplished with reactive, phosphine-containing chemical modification reagents; alternatively, fluorescent dyes and affinity tags such as biotin are available ready-made in phosphine-activated form,Better than Azide-Alkyne Click Chemistry Although azide-alkyne (“click“) chemistry uses the same azide component as the azide-phosphine (Staudinger) chemistry, it requires special copper-containing reaction buffers that have damaging effects on cellular components.,Azide-Phosphine (Staudinger Ligation),/browse.cfm?fldID=020305,Site-Specific Protein Labeling and Protein Interaction Detection with Biotin Ligase,Labe