冯喜增《结构生物学》第九讲-2-圆二色光谱及其应用.pptVIP

圆二色光谱Circular Dichroism (CD);1.什么是圆二色谱?;What is a Light? ;UV;UV;?;;;X;;X;X;频率相同,方向相反 振幅相同、相位不同;? ;? ; 合成光特性;椭圆偏振光;振动方程;圆二色性与振幅 ;;旋光色散与位相;平均残基旋光度 [m]l the mean residue rotation;CD原理;平面偏振光 (Plane polarized light);旋光色散(Optical rotatory dispersion);旋光度;旋光色散;圆偏振光(Circular polarized light);圆偏振光;圆二色性(circular dichroism, CD);;圆二色性的表示;旋光色散与圆二色性;叠加原理;;;旋光色散与圆二色相互转换;圆二色仪原理;;Instrument;;圆二色谱的噪音;蛋白质的CD谱;The peptide bond is inherently asymmetric is always optically active;;Near UV CD spectrum of Lysozyme;蛋白质的CD谱;Far UV CD spectra of poly-L-Lys;;;;估算蛋白质?螺旋含量;If we measure the CD signal for a protein of unknown structure we can find its proportion of 2ndry structures;Example fit: myoglobin;Example fit (2): GCN4-p1;CD signals for same secondary structure can vary (a bit) with environment;Recommended Methods;Best fitting procedures use many different proteins for standard spectra;Determination of Protein Concentration;Limitations of CD secondary structure analysis;CD signal of a protein depends on its 2ndary structure;从CD谱分析蛋白质的结构类型;CD is very useful for looking at membrane proteins;Instrumentation: synchrotron–based;Applications of CD in structural biology;CD application;;克山病探究-硒缺乏;烟草花叶病毒侵染膜蛋白;;Unfolding/folding;Conformational varies;;; Near UV spectra: to investigate the tertiary structure;振动圆二色谱;Protein Concentration: 0.5 mg/ml Cell Path Length: 0.5 mm Stabilizers (Metal ions, etc.): minimum Buffer Concentration : 5 mM or as low as possible while maintaining protein stability;参考文献;What is meant by “far UV” light? When CD is applied to proteins what is the main chromophore (absorbing group) that is looked at? What wavelength range does it absorb in? You are measuring the CD spectrum of a protein using a 1mm path length cell. You find that the signal is weak because the concentration of the protein is lower than you were initially told. How could you overcome this problem – and what do you have to be careful of. Does the CD spectrum of a protein give most information about the