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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,10,100 Kcal/mol,Bonds break,Light used for vision and photosynthesis,Electromagnetic spectrum,Nuclear spin,vibrations,Electronic transitions,The longer the wavelength,the lower the energy.,基本原理1-Lambert Law,Th
2、e 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2-Beers Law,The amount of light absorbed is proportional to the number of molecules of
3、the chromophore through which the light passes. k=c,Deviations from the Beer-Lambert law,强吸收 高浓度,噪声与误差,等吸收点(Isosbestic point),等吸收点常作为体系中只有两种成分(状态)的指示 可作为参比波长 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,Wavelength,Absorbance,Concentration,A,B,A,B,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,
4、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.cm 190nm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,1mM,0.1mM,0.1mM,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/prot
5、param.html,Tyr的吸收谱与pH有关,pH titration can be used to determine - whether Tyr is internal or external - polarity of environment,max at pH 6: 274 nm max at pH 13: 295 nm,环境极性的影响,Blue shift of spectra in polar solvent Weaker absorption of Tyr in polar solvent,蛋白质构象变化研究,Absorption spectra of Poly-L-Lys H
6、Cl: 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,0,3,3,0,The Franck-Condon principle: transitions are vertical in both absorption and emission,The Franck-Condon factor is the same for absorption and fluorescence,00,01,10,03,06,30,
7、60,Exceptions:- very long lived S1 state : emission occurs from 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 (1-10-cl) F 若cl很小，则近似： IF = I0(cl)F Inner filter effect,Inner filter effect,所以，实验中，A,EX0.1,Raman and Rayleigh Scattering,Two potential sources
8、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 wave
9、length. 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O-H收缩(3300cm-1): 1/ RA = 1/ EX 0.00033,Environmental Sensitivity,Fluorophores can be affected by a large number
10、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 fl
11、uorescence,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 spectrum,Excitation spectrum should correspon
12、d 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.,Experiments,Spectral shifts: Intrinsic, Extrinsic Fluorescence Fluoresc
13、ence quenching: Internal, External Fluorescence Resonance Energy Transfer (FRET) Rotation of molecules: fluorescence anisotropy Fluorescence lifetime ,Protein intrinsic fluorescence,Trp fluorescence can be selectively excited at 295-305 nm. (to avoid excitation of Tyr),Trp is the dominant intrinsic
14、fluorophore in proteins,Trp 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 rin
15、g. 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 shifts,Gas phase,m,m*,solvent,Solvent can affect the ground state and excited state molecules causing spectral shiftE
16、xample: H bonding to tryptophan. Changes its absorption by about 10 nm,Fluorescence of tryptophan depends upon the dipolar nature of the solvent,Ca-parvalbumin: tryptophan is buried, 305 nm,Ca-free parvalbumin: tryptophan is exposed to solvent,Tryptophan in various solvents: hexane, trehalose glass, glycerol, water,
