Transcript 圆二色原理和应用报告()

圆二色谱Circular Dichroism (CD)
Application
圆二色光谱仪通过测量生物大分子的圆二色光
谱从而得到生物大分子的二级结构。
可应用于：蛋白质折叠﹑蛋白质构象研究,
DNA/RNA反应, 酶动力学, 光学活性物质纯度测
量, 药物定量分析。天然有机化学与立体有机化
学, 物理化学, 生物化学与宏观大分子, 金属络合
物, 聚合物化学等相关的科学研究。
构象
确定蛋白质构象最准确的方法是x-射线晶体衍
射，但对结构复杂、柔性的生物大分子蛋白质
来说，得到所需的晶体结构较为困难。二维、
多维核磁共振技术能测出溶液状态下较小蛋白
质的构象，可是对分子量较大的蛋白质的计算
处理非常复杂。
圆二色光谱：研究稀溶液中蛋白质构象，快速、
简单、较准确
CD is very useful for looking at membrane proteins
 Membrane proteins are difficult to study.
 Crystallography difficult - need to use detergents
Often even when structure obtained:
Q- is it the same as lipid?
 CD ideal can do spectra of protein in lipid vesicles.
 We will look at Staphylococcal a-hemolysin as an
example
主要内容
CD原理
蛋白质CD谱
CD实验要点
CD原理
圆二色性(circular dichroism, CD)
当平面偏振光通过具有旋光活性的介质时，
由于介质中同一种旋光活性分子存在手性不同
的两种构型，故它们对平面偏振光所分解成的
右旋和左旋圆偏振光吸收不同，从而产生圆二
色性．
圆二色性的表示
椭圆度，摩尔椭圆度[]
=2.303(AL – AR)/4
[] = 3298(L - R)3300 (L - R)
在蛋白质研究中，
常用平均残基摩尔椭圆度
圆二色仪原理
蛋白质的CD谱
蛋白质的光学活性
The peptide bond is inherently asymmetric & is always optically active
蛋白质的CD谱
 CD spectra in the far UV region (180 nm –
250 nm) probes the secondary structures
of proteins.
 CD spectra in the near UV region (~250
and ~ 350) monitors the side chain tertiary
structures of proteins.
Near UV CD spectrum
蛋白质中芳香氨基酸残基，如色氨酸(Trp)、酪氨酸
(Tyr)、苯丙氨酸(Phe)及二硫键处于不对称微环境时，
在近紫外区250～320 nm，表现出CD信号。
Phe残基: 255、261和268 nm附近；Tyr残基:277 nm左
右；而在279、284和291 nm是Trp残基的信息；二硫键
的变化信息反映在整个近紫外CD谱上。
近紫外CD谱可作为一种灵敏的光谱探针，反映Trp、
Tyr和Phe及二硫键所处微环境的扰动，能用来研究蛋
白质三级结构精细变化。
Near UV CD spectrum of Lysozyme
100

0
-100
-200
260
270
280
290
nm
300
310
Main CD features of protein 2ndary structures
- band (nm)
+ band (nm)
α-helix
222
208
192
β-sheet
216
195
β-turn
220-230 (weak)
180-190 (strong)
205
polypro II helix
190
210-230 weak
Random coil
200
212
Far UV CD spectra of poly-L-Lys
CD signals for same secondary structure can
vary (a bit) with environment
 But on a coiled-coil breaks down helical
 Can see this by looking
dimer to single helices
at the effect of trifluoroethanol (TFE)
on a coiled-coil similar to
Effect of 50% TFE on a coiled-coil
GCN4-p1
 TFE induces helicity in all peptides
0
TM-36 aqueous
TM-36 + TFE
-5
Effect of 50% TFE on a monomeric peptide
MRE
-10
-15
-20
0
peptide in water
peptide in 50% TFE
-25
-5
-30
TFE
-10
MRE
-35
200
-15
TFE
-20
210
220
230
240
wavelength in nm
 Although 2ndry structure same
CD changes
-25
-30
-35
200
210
220
230
wavelength in nm
240
Lau, Taneja and Hodges (1984)
J.Biol.Chem. 259:13253-13261
Best fitting procedures use many different proteins for
standard spectra
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There are many different algorithms.
All rely on using up to 20 CD spectra of proteins of known structure.
By mixing these together a fit spectra is obtained for an unknown.
For full details see
Dichroweb: the online CD analysis tool
www.cryst.bbk.ac.uk/cdweb/html/
 Can generally get accuracies of
0.97 for helices,
0.75 for beta sheet,
0.50 for turns, and
0.89 for other structure types
(Manavalan & Johnson, 1987, Anal. Biochem. 167, 76-85).
估算蛋白质a螺旋含量
仅适合a含量较高的蛋白质！
*Yang算法
Limitations of CD secondary structure
analysis
 The simple deconvolution of a CD spectrum into 4 or 5
components which do not vary from one protein to another is a
gross over-simplification.
 The reference CD spectra corresponding to 100% helix, sheet,
turn etc are not directly applicable to proteins which contain
short sections of the various structures e.g. The CD of an αhelix is known to increase with increasing helix length, CD of βsheets are very sensitive to environment & geometry.
 Far UV curves (>275nm) can contain contributions from
aromatic amino-acids, in practice CD is measured at
wavelengths below this.
 The shapes of far UV CD curves depend on tertiary as well as
secondary structure.
蛋白的三级结构
1976年，Levitt和Chothia曾在Nature上报道，规则蛋白质
的三级结构模型可分为4类
(1) 全α型，以仅α-螺旋结构为主，其分量大于40％ ，而
β-折叠的分量小于5％
(2) 全β型，以β-折叠这种结构为主，其分量大于40％ ，而
仅一螺旋的分量小于5％ ；
(3) α+β型，α螺旋及β-叠折分量都大于15％ ，这两种结构
在空间上是分离的，且超过60％的折叠链是反平行排列；
(4) α/β型， α-螺旋和B-折叠含量都大于15％ ，它们在空间
上是相间的，且超过60％的折叠链平行排列。
CD signal of a protein depends on its 2ndary structure
—— chymotrypsin (all b)
—— lysozyme (a + b)
—— triosephosphate isomerase(a/b)
—— myoglobin (all a)
从CD谱分析蛋白质的结构类型
(Venyaminov & Vassilenko)DEF_CLAS.EXE:
对全a、 a /b和变性蛋白质的准确度为100%，
对a + b的准确度为85%，
对全b的准确度为75%。
对多肽的判断较差！
CD实验要点
Determination of Protein Concentration
精确的方法有：
1 定量氨基酸分析；
2 用缩二脲方法测量多肽骨架浓度 或测氮元素
的浓度 ；
3 在完全变性条件下测芳香氨基酸残基的吸收，
来确定蛋白质的准确浓度.
 Not Acceptable:
1. Bradford Method.
2. Lowry Method.
3. Absorbance at 280 and/or 260 nm.
Nitrogen flushing
Flushing the optics with dry nitrogen is a must:
 Xe lamp has a quartz envelope, so if operated in
air it’ll develop a lot of ozone, harmful for the
mirrors
 below 195nm oxygen will absorb radiation
HT plot
 The HT plot is very important, since readings above 600650V mean that not enough light is reaching the
detector so a sample dilution or the use of shorter path cell
are required.
 Furthermore the HT plot is in realty a single beam spectra
of our sample, since there is a direct relation between HT
and sample absorbance. By data manipulation HT
conversion into absorbance and buffer baseline subtraction
is possible. Alternatively single beam absorbance scale can
be used already in CH2 during data collection, loosing
however a bit the alerting functions of this channel.
Bandwidth (SBW) selection
 Setting of slits should be as large as possible (to decrease
noise level), but compatible to the natural bandwidth
(NBW) of the bands to be scanned.
 As a rule SBW should be kept at least 1/10 of the NBW,
otherwise the band will be distorted.
 If NBW is not known a series of fast survey spectra at
different SBW will help proper selection. Trade in of
accuracy versus sensitivity (i.e. the use of larger than
theoretical SBW) is occasionally required.
 2 nm in the far UV region
 1 nm in the aromatic region (where fine structures may be
present), optimal band-pass (as large as possible, but not
loosing information) can be determined after a trial
Number of data point
 data pitch, i.e. number of data points per nm, will
not directly influence the noise level. However if
post run further data processing will be applied to
reduce the noise, it’s advisable to collect as many
data points as possible to increase the efficiency of
the post run filtering algorithm
Accumulation
 another way to improve S/N is to average more spectra.
Here too the S/N will improve with the square root of the
number of accumulations.
 Averaging is very effective since it compensates short term
random noise, but it’ll not compensate long term drifts
(mainly of thermal origin). So if long accumulations are
used we recommend a suitable long warm-up of the system
and/or the use of a sample alternator (to collect
sequentially sample and blank and average their subtracted
values).
 For long overnight accumulations it’s essential that room
temperature is well kept stable.
Sample concentration and cell pathlength
 A good suggestion is to run in advance an
absorption UV-VIS spectra.
 CD spectroscopy calls for same
requirements as UV-VIS: best S/N is
obtained with absorbance level in the range
0.6 to 1.2. It’s usually difficult to get proper
data when absorbance (of sample + solvent)
is over 2 O.D.
Typical Conditions for protein CD
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Protein Concentration: 0.2 mg/ml
Cell Path Length: 1 mm
Volume 350 ml
Need very little sample 0.1 mg
Concentration reasonable
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration : 5 mM or as low as possible while
maintaining protein stability
溶剂的吸收!!!
Buffer Systems for CD Analyses
 Acceptable:
1. Potassium Phosphate with KF, K2SO4 or
(NH4)2SO4 as the salt.
2. Hepes, 2mM.
3. Ammonium acetate, 10mM.
 Avoid: Tris; NaCl; Anything optical active, e.g.
Glutamate
Summary
 CD is a useful method for looking at secondary structures of
proteins and peptides.
 CD is based on measuring a very small difference between two
large signals must be done carefully
 the Abs must be reasonable max between ~0.6 and ~1.2.
 Quarts cells path lengths between 0.0001 cm and 10 cm. 1cm and
0.1 cm common
 have to be careful with buffers TRIS bad - high UV abs
 Measure cell base line with solvent
 Then sample with same cell inserted same way around
 Turbidity kills - filter solutions
 Everything has to be clean
 For accurate 2ndry structure estimation must know
concentration of sample
核酸的CD信息
B-Z?
Or
Z-B？
建议浓度：在吸光值0.2-0.8时浓度的5-10倍
圆二色谱在糖类化合物结构分析中的应用
 碳水化合物的结构决定CD的强度和形状，而从CD获
得的构象信息的多少取决于样品结构的复杂程度；
 对那些具有较好重复系列的糖类化合物而言，CD能提
供更为可靠的空间结构信息。
 对那些结构比较复杂的糖类来说，即使不能直接测定
糖类化合物的绝对构象，利用一些经验规则，例如，
CD可以用来推断糖类是否具有gt构象，而且CD可以作
为探针来测定糖类化合物构象的变化，如胶体-溶液或
无序-有序的转变过程。
THANKS!