紫外-荧光在蛋白质分析中的应用9培训教材.ppt

单击此处编辑母版标题样式单击此处编辑母版副标题样式*1紫外与荧光光谱在蛋白质研究中的应用 主要内容UV/vis基础、实验要点、应用Fluorescence基础、实验要点、常规荧光、淬灭、荧光共振能量传递(FRET)谱学方法基本原理1Lambert LawThe fraction of light absorbed by a transparent medium is independent of the incident intensity, and each successive layer of the medium absorbs an equal fraction of the light passing through it. Log10(I0/I)=kl基本原理2Beers LawThe amount of light absorbed is proportional to the number of molecules of the chromophore through which the light passes. k=cDeviations from the Beer-Lambert law强吸收高浓度噪声与误差等吸收点(Isosbestic point)v 等吸收点常作为体系中只有两种成分(状态)的指示v 可作为参比波长v 若有等吸收点,则不需要为为每个谱作基线单光路双光路双波长二极管阵列检测器二极管阵列光谱仪动力学实验WavelengthAbsorbanceConcentrationABAB波长选择常规吸收光谱实验要点样品准备波长选择池子选择扫描速度选择带宽选择石英、玻璃、塑料慢快若样品不稳定若噪声大，分辨率低等窄宽若噪声大合适的浓度，正确的参比紫外/可见光谱在蛋白质研究中的应用浓度测定构象变化研究相互作用研究蛋白质主链的紫外吸收肽键在250 nm的远紫外区有较大吸收蛋白质浓度测定190nm 10000 l/mol.cm190nm比280nm大100倍但溶剂吸收也较大溶剂的吸收蛋白质中氨基酸的紫外吸收光谱1mM0.1mM0.1mM二硫键在250 nm附近有弱吸收确定蛋白质的消光系数可以根据蛋白质序列预测蛋白质的消光系数 ProtParam:http:/cn.expasy.org/tools/protparam.htmlTyr的吸收谱与pH有关pH titration can be used to determine whether Tyr is internal or external polarity of environmentmax at pH 6: 274 nmmax at pH 13: 295 nm环境极性的影响Blue shift of spectra in polar solventWeaker absorption of Tyr in polar solvent蛋白质构象变化研究Absorption spectra of PolyLLys HCl: random coil at pH 6.0, 25C(bold);Helix at pH 10.8, 25C(dotted); strand at pH 10.8, 52C(dashed).蛋白质折叠/变性研究荧光光谱03300022The FranckCondon principle: transitions are vertical in both absorption and emissionThe FranckCondon factor is the same for absorption and fluorescence00011003063060Exceptions: very long lived S1 state : emission occursfrom a different geometry. Reactions from the excited state.量子产率F = 发射的光子数/吸收的光子数荧光强度IF = I0 (110cl) F 若cl很小，则近似：IF = I0(cl)F Inner filter effect Inner filter effect所以，实验中，A,EX0.1Raman and Rayleigh ScatteringTwo potential sources of background radiation are Raman and Rayleigh scattering. Both of these phenomena arise due to vibrational changes induced in molecules by incident radiation. Both can also be described as scattered light.The Raman lines are of different frequency from the incident light and usually of longer wavelength. Rayleigh radiation is scattered light of the same frequency as the incident light.Rayleigh Scattering强度与r6/ 4成正比不能通过减空白来消除选择合适的激发波长从比激发波长大(10nm)处开始收谱减少带宽Raman scattering因水中OH收缩(3300cm1):1/ RA = 1/ EX 0.00033Environmental SensitivityFluorophores can be affected by a large number of environmental factors including such parameters as pH, ionic strength, non-covalent interactions, light intensity, temperature and so on. Both the excitation and emission wavelengths and the quantum yield can all be changed by environmental factors.温度荧光一般随温度上升而减弱荧光实验需要恒温2 ways of measuring fluorescence Emission spectrum excitation constant, measure fluorescence intensity of emission against , I.e. spectrum of emitted light. Excitation spectrum measure fluorescence intensity at different excitation , similar to absorption spectra.Excitation spectrumExcitation spectrum should correspond colsely with the absorption spectrum of the molecule that is responsible for the fluorescence.Excitation spectrum reveals whether the sample is homogeneous, and whether all fluorescence features result from a single molecule.ExperimentsSpectral shifts: Intrinsic, Extrinsic FluorescenceFluorescence quenching: Internal, ExternalFluorescence Resonance Energy Transfer (FRET)Rotation of molecules: fluorescence anisotropyFluorescence lifetime.Protein intrinsic fluorescenceTrp fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr)Trp is the dominant intrinsic fluorophore in proteinsTrp fluorescence is very sensitive to its local environment It is possible to see changes in emission spectra in response to conformational changes, subunit association, substrate binding, denaturation, and anything that affects the local environment surronding the indole ring. Also, Trp appears to be uniquely sensitive to collisional quenching, either by externally added quenchers, or by nearby groups in the protein.Local electric fields cause spectral shiftsGas phasemm*solventSolvent can affect the ground state and excited state molecules causing spectral shiftExample: H bonding to tryptophan. Changes its absorption by about 10 nmFluorescence of tryptophan depends upon the dipolar nature of the solventCaparvalbumin: tryptophan is buried, 305 nmCafree parvalbumin: tryptophan is exposed to solvent Tryptophan in various solvents: hexane, trehalose glass, glycerol, waterFluorescence spectral shifts can be very largeTyr fluorescence 若蛋白质只含TYR,不含TRP：蛋白质变性后，荧光强度明显增强。

GFP is isolated from the Pacific jellyfish Aequorea victoria and now plays central roles in biochemistry and cell biology due to its widespread use as an in vivo reporter of gene expression, cell lineage, protein protein interactions and protein trafficking GFP can be fused to another protein either N or Cterminally. This is due to the fact that both termini of GFP appear rather flexible on the s。