《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 Localization of Molecules with Fluorescence MicroscopyMicroscopy Image from Molecular Probes (Invitrogen): Fluorescence Applications in BiologyFluorescence 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 MoleculesExamples 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 ChromophoresAffinity LabelsSpin LabelsCatalystsCrosslinkersPolymers EnzymesNanocrystalsMRI Contrast AgentsMaterial Surfaces Chemical Reaction Radiolabels Common Examples of Protein BioconjugatesCommon 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 p中性条件 p在氨基的存在下也可选择性与巯基反应 p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 p 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 p比碘乙酰胺选择性更高 p中性条件pH 7, pH 高于8水解成不反应的顺丁烯二酸 p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 nMediate the formation of amide linkages between a carboxylate and an amine, or phosphoramidate linkages between a phosphate and an amine. nMost popular - two protein molecules, a peptide and a protein, oligonucleotides and proteins, etc. n水溶/非水溶 - 生物大分子/多肽、有机合成 nEDC, EDC plus Sulfo-NHS, CMC, DCC, DIC EDC (1-(3-二甲氨基丙基)-3-乙基碳二亚胺) n水溶性好，多余试剂、副产物易去除 n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 n 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 Biological Labeling with Green Fluorescent Protein (GFP)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. 1.Protein folding 2.Cyclization 3.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 p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） p六组氨酸(His6 ) 识别结合 Ni2+：次氮基三乙酸（Ni2+:NTA) p 蛋白质C端、N端、或肽链中间 p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. 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. FeaturesBenefits Chemoselective reactivity Virtually no side reactions with other functional groups Detect and enrich without contamination Accurate and representative biological observations Compatible with bioorthogonal metabolic labeling Terminal azido functional groups not found in living systems Azido derivatives of biological molecules are well tolerated High reaction efficiencyClose to 100% reaction efficiency in chemical model systems Ease of use Two-reagent system is easier to use and troubleshoot than alternative copper-catalyzed click chemistry (5 different reagents) Biological-sample compatibility Reactions occur in aqueous solutions at physiological pH with no known side reactions with buffers/detergents No risk for toxicity and protein oxidation because of the absence of Cu(I) Allows probe switching Azido-labeled molecules can be derivatized with wide variety of phosphine probes Azide-Phosphine (Staudinger Ligation) /browse.cfm?fldID=020305 Site-Specific Protein Labeling and Protein Interaction Detection with Biotin Ligase L