同步辐射圆二色光谱技术

同步辐射圆二色光谱技术中国科技大学国家同步辐射2008-041同步辐射圆二色光谱技术1，圆二色光谱概念及其意义2，基本原理3，同步辐射圆二色光谱的优势4，同步辐射圆二色光谱测量方法5，国家同步辐射

圆二色光谱仪器简介2光的偏振性3光的偏振性光的偏振性5光的偏振性光的偏振性7Plane-polarized

waves

in

an

absorbing

medium8Circularly

polarized

waves

in

an

absorbing

medium9Plane-polarized

waves

in

a

refracting

medium10Circularly

polarized

waves

in

a

refracting

medium11Plane-polarized

waves

in

amedium

showing

circular

dichroismcircular

dichroism

makes

plane-polarized

lighliptically

1po2lar13Plane-polarized

waves

in

amedium

showing

circular

birefringencecircular

birefringence

rotates

the

plane

of

polarization

of

plane-polarized

light.Plane-polarized

waves

in

a

medium

showing

both14circular

dichroism

and

circular

birefringenceWhat

is

Circular

Dichroism

(CD)物质吸收圆偏振光时，如

果对左旋和右旋圆偏振光

的吸收表现出一定的差异，则称该物质具有圆二色性。1516圆二色光谱的意义生物大分子如蛋白质和DNA是由光学活性基团组成，并且它们可以采取不同的三维结构，因此，每一种分子都有特征的CD谱。研究蛋白质和DNA最有用的光谱区是紫外和真空紫外区（160

to

300

nm)，因为来自蛋白质的缩氨酸主链和侧链以及DNA的嘌呤和嘧啶的电子跃迁就发生在这一光谱能量区。氨基酸是蛋白质的单体。天然存在于蛋白质中的氨基酸共有20种。氨基酸由肽键相连形成多肽链。蛋白质结构17蛋白质结构的层次一级结构氨基酸序列二级结构主要由氢键稳固的局部构象，如-helix,-sheet等三级结构三维构象四级结构多个多肽链的组合18蛋白质的结构CD19XRDNMR二级结构与圆二色谱20二级结构与圆二色谱-helix:

19x

nm

(+)208nm,

222nm

(-)21-sheet:

195nm

(+)antiparallel:

red

shiftparallel:

blue

shift215-7nm

(-)Unordered

structure:(-)

below

200nm(+)

218nm

weak

band-turn:180-190nm

(-)200-205nm

(+)225nm

(+)

band,

weakredshift,s[

]

,[

][

]

,t[

]

,圆二色光谱的解析Circular

dichroism

spectra

oftriosephosphate

isomerase(H:0.52,

S:0.14,

T:0.11,O:0.23),

hen

egg

lysozyme(H:0.36,

S:0.09,

T:0.32,O:0.23),

myoglobin

(H:0.78,S:0.0,

T:0.12,

O:0.10),

andchymotrypsin

(H:0.10,

S:0.34,T:0.20,

O:0.36).22[

]

f

[

]

,

fs

[

]

,s

ft

[

]

,t

f

[

],

...Cartoon

drawings

of:

A)

triosephosphate

isomerase

(H:0.52,

S:0.14,

T:0.11,

O:0.23);

B)henegg

lysozyme

(H:0.36,

S:0.09,

T:0.32,

O:0.23);

C)

myoglobin

(H:0.78,

S:0.0,

T:0.12,O:0.10);

and

D)

chymotrypsin

(H:0.10,

S:0.34,

T:0.20,

O:0.36).

Secondary

structuresarecolor

codedred:helix.

green:strand,

andyellow:other.23圆二色光谱的解析ysing

hydro2ge4n

bonds.CCAConvex

constraintalgorithm

(Perczel

et

al)CONTINImplementation

of

ridge

regression

algorithm.

Several

versions

available...

the

latest

contin/ll

incorporates

thelocally

linearised

model

of

van

Stokkum

et

al.CDFITLinear

least

squares

fit

method

configured

for

JASCO

formatted

files.CDPROSuite

of

programmes

including

the

latest

versions

of

CONTIN

SELCON

and

CDSSTR.CDSSTRModified

version

of

the

variable

selection

programme

by

Johnson

et

al

(Varslc)

to

utilisea

numberof

differentreference

datasets.DichroProt/Antheprotysisdesigned

for

windows

but

with

sourcecodes

availableforSuite

of

programmes

for

protein

structureVarslc.K2DNeural

network

approachfrom

Andrade

et

alBLinear

combination

least

squares

method

available

with

CCASELCONThe

self-consistent

method

(Sreerama

&

Woody).

Several

verions

available.VARSLCThe

original

version

of

the

variable

selection

method

by

Johnson

et

al.XTLSSTRA

programme

from

Johnson

to

obtain

structure

fractions

from

PDB

files

by解谱程序25圆二色光谱的优点简单、快捷样品不需要做过多准备在溶液状态下测量相对少的样品用量、低浓度微秒时间分辨适用于任何尺寸的大分子样品26CD

vs

XRD（NMR)CD约30分钟XRD、NMR几天——几个月：筛选、优化结晶条件，得到蛋白质晶体获得衍射图、确定相位建立模型并优化，最终获得结构信息27测定不能结晶的蛋白质的二级结构研究结合在蛋白质二级结构上的药物的功能动态过程如蛋白质折叠的研究研究环境对蛋白质结构的影响研究膜蛋白的二级及超二级结构研究基质引起的构型变化碳水化合物构型研究研究蛋白质-蛋白质及蛋白质-核酸的相互作用识别折叠状态圆二色光谱的意义基本原理——数学描述

AClI

I010

I010A

AL

AR

LCl

RCl

028圆二色定量描述：对左旋和右旋圆偏振光吸收度的差异y’y

L

=

tg-1[(|ER

-

EL|)/(ER

+

EL)]圆二色性的描述——椭圆率θElErxRx’基本原理——数学描述29旋光色散与圆二色性1

光在介质中的Rate:

c

/

n

,

(n

折射率) n

=fdirection光强I:A=lg(I0

/I)=Cl

f2

ORD(optical

rotatory

dispersion)?圆双折射(Circular

birefringence):nl导致左、右旋圆偏振光相位不同nrChiral

compd.Optical

active

object

L

nn

=

nl

-

nr

f

(

)偏转角度(旋光度)与波长所建立起的函数关系3031比旋光度

[]

(the

specific

rotation)摩尔比旋光度

[]

(the

molar

rotation)平均残基旋光度

[m]

(the

mean

residue

rotation)[]

=

/Cl[]

[]

[Mw/100][m]

=

[]

[MRw/100]单位:(*****)C

:

g/mLl

:

dmMw:

molecular

weightMRw:

the

mean

residue

Mw旋光色散与圆二色性旋光色散与圆二色性3

CD

(circular

dichroism)光强不同、或左右旋圆偏振光的吸收率或消光度不同A=

Al

-

Ar

,

=

l

-

r

=

A

/

ClA

or

f

(

)

33.00

A

Cl

(the

specific

ellipticity比椭圆率)[

]

Cw

l[

]

Mw

[

]/100

(the

molar

ellipticity摩尔椭圆率)[

]MRw

MRw

[

]/100

(the

mean

residue

ellipticity平均残基椭圆率)旋光色散与圆二色性[

]

100

Cl

=

3300

C:摩尔浓度

l:光程(cm)[

]

[deg.

mol-1

.

cm-1

]

or

[deg

.

cm2

.

dmol-1

]4

relationship

between

CD

and

ORD

and

Cotton

effectLightnl

,

nrxRx'Ly

y'

[]

f

(

)[

]

f(

)ORDCDKronig-

Kramerstransition33基本原理——数学描述2I

E2I0

E0

e2.303A

/

2E

E0

e2.303

AR

/

2

e2.303

AL

/

2tg

e2.303

AR

/

2

e2.303

AL

/

2R

A

)

/

4)L

tgh(2.303(

Atg

tgh[

]

2.303(

AL

AR

)434基本原理——数学描述I

()

I

()

I

()

I

()10

Al

(

)

I

()10

AR

(

)L

R

0

0

I

()10(

A(

)A(

)/

2)

I

()10(

A(

)A(

)

/

2)0

0A()

(AL

()

AR

())

/

2A()

AL

()

AR

()A()

A()

A(

)I

()

I0

()10

A()

I

()A()A()

I

()

I

()

10635常规圆二色光谱仪的组成锁相放大器光弹调制器器36同步辐射优越性同步辐射优越性同步辐射优越性真空紫外常规连续谱光源3940同步辐射优越性光

源波长范围（nm）光谱辐射峰值功率（mW/mm2.sr.nm）氢灯（1KW）165——2500.1(在230nm)氙灯150——2250.01(在170nm)氪灯125——1650.001(在150nm)同步辐射X-ray——IR1同步辐射优越性41同步辐射园二色光谱的优越性42同步辐射园二色光谱的优越性同步辐射园二色光谱的优越性44同步辐射优越性弯铁弯铁产生的同步辐射光的偏振特性：在 轨道平面内是线偏振光，在轨道平面上方及下方是手性相反的椭圆偏振光45同步辐射优越性水平与垂直偏振分量的归一化强度与波长、观察角的关系46在这个波长范围，由于有几种透光晶体材料，获得圆偏振光的方法有些类似常规的做法，即首先获得线偏振光，然后再通过光弹调制器得到圆偏振光。所不同的是，不再需要晶体线偏振器来获得线偏振光，而是直接利用同步辐射光的偏振特性。紫外-真空紫外区（120nm≤λ≤300nm）同步辐射圆偏振光的获得弯铁利用偏振挡光板获得线偏振同步辐射光，然后由光弹调制器转换为圆偏振光（用于

国家同步辐射光源NSLS）球面光栅球面反射镜狭

缝偏振挡光板光弹调制器47同步辐射圆偏振光的获得弯铁狭

缝光弹调制器利用镀金反射镜获得线偏振同步辐射光，然后由光弹调制器转换为圆偏振光镀金反射镜光栅48Major

requirements

for

VUV

Circular

DichroismBeamline

atNSRLWavelength

range:

120nm-300

nm49Resolution:High

flux

:0.3nm~1013

ph/sHigh

degree

of

linear

polarizationOptical

Layout

of

the

CD

BeamlineBendingMagnetElectronBeamCylindrical

GratingCylindrical

mirrorPolarization

plateslit

PEM3500mm1900mm220Sample50DetectorPrefocusing

MirrorGratingRefocusing

mirrorFigureCylindricalCylindricalToroidalDimension

(mm3)150x40x25150x40x2550x30x25Parameter(mm)r=2837.1R=2555.19R=341.08,

ρ=154.28Blank

MaterialFused

SilicaFused

SilicaFused

SilicaSurface

CoatingLine

DensityAl+MgF2Al+MgF2600l/mmAl+MgF2Parameters

of

the

Optical

Elements51Predicted

Characteristics-resolution1004Å997Å1000ÅSpot

diagram

on

slit（600g/mm、1000Å）52Predicted

Characteristics-total

flux

atsampleReflectivityof

the

mirror:

0.65Efficiency

of

the

grating:

0.1Resolution:

0.3nm53Predicted

Characteristics-Image

onsample54Predicted

Characteristics-Linear

Polarization

degreeVertical

aperture:

3.5mrad5556Photon

flux~1013photons/sec(200nm,0.3nm,300mA)Wavelength

Range120-300nmResolution0.3-3nmLinear

Polarization>70%(depending

on

vertical

aperture)Image

size

at

Sample(h×v)3×2

mm

for

CD

or

2×1mm(for

PA)Summary

ofthe

beamlineSketch

for

the

experimental

stationWavelength

ControlComputerWavelengthControlBeamlinePEM

ControlPEMVoltageCompareLock-inAc

Signaldc

SignalPMTHigh

VoltageSampleReference

frequency5758Drawing

of

the

BeamlinePicture

of

the

beamline59Picture

of

the

PEM-90

(Calcium

Fluoride)60Examples-Drug

discovery

and

developmentThe

high

precision

of

SRCD

measurements

allows

the

identificationof

verysmall

changes

in

secondary

structure.

Combined

with

the

speed

and

simplicityof

the

measurements,

this

makes

SRCD

a

suitable

techniquefor

the

screeningof

potential

drug

compounds

for

their

effect

on

protein

secondary

structure61Examples-Investigation

of

protein

foldingExamples-Polysaccharide

structure

for

the

development

of

foodand

pharmaceuticals63Examples-Amyloid

diseases4Examples-Materials

research

and

development6566SRS,

Daresbury;

UKNSLS,

Brookhaven

National

Laboratory,

USASTRID,

Aarhus

DenmarkHiSOR,

Hiroshima,

JapanBessy

II,

GermanyELSA,

Bonn,

GermanySRCD

today6768锁相放大器及其工作原理1962年第一台仪器问世，发现俄歇效应。据统计，已在几百种场合中得到应用。

在弱信号探测仪器中锁定放大器是一个

非常重要的品种。信噪比可低达10-5。BW=0.0004Hz（相当于Q值=108）。两个随机过程的相关性对两个信号x(t)和y(t)，定义相关函数–

互相关函数Rxy

,Ryx

：T

y(t

)

x(t

)dtT

/

2T

/

21yxR

(

)

TLim69T

T

/2T

/2x(t

)

y(t

)dt1TRxy

(

)Lim随机过程的相关性–自相关函数：70T

)dtR(

)

T

/

2T

/

2x(t)x(t1TLim白噪声，自相关函数=0自相关检测积分器乘法器延迟S)Rss(τ)参考通道信号通道71自相关检测设有信号S(t)，通过如上图所示的功能器件，则：

)

(

ST

T

/21

T

/2R(

)lim

[T(

)

(

)][

(

StSnttS[S(t)t(n))n(tT

T

/21

T

/2limTRSS

(

)RnS

(

)RSn

(

)Rnn

(

)RSS

(

)Rnn

(

)假定噪声的平均值等于零7273正弦波与噪声之和的自相关函数SignalS(t)V

cos(wt)21nn

R

(

)

)

VcosCorrelated function

R(积分器乘法器S)Rss(τ)参考通道信号通道延迟互相关检测74互相关检测设有信号S(t)，通过如上图所示的功能器件，则：tS

StT

T

2/T

2/21())(][1R

)(

limT21

2S

SR

(

)RnS

(

)2((t)S(ST

T

/2T

/2211limT1

2S

SR

(

)75内□前置被测信号滤波器主放灵敏度乘法器T积分器DC放大输出参考信号产生振荡相位乘法器的工作过程V+-接差动积分器信号地参考信号77被测信号积分器工作过程低通滤波器及特性>f78100%乘法器（Phase-Sensitive

detector）输出Vpsd

=

Vsigsin(rt

+

sig)

VLsin(Lt

+

ref)=

1/2

VsigVLcos([r

-

L]t

+

sig

-

ref)-1/2

VsigVLcos([r

+

L]t

+

sig

+

ref)当r=L低通滤波器Vpsd

=

1/2

VsigVLcos(sig

-

ref)7980信号：10nV

sinewave

at

10

kHz1,

低噪声放大器：

about

5

nV/Hz

of

input

noise，bandwidth

100

kHz

，gain

1000output

：

10µV

of

signal

(10

nV

x

1000)1.6

mV

of

broadband

noise

(5

nV/Hz

x

100

kHz

x

1000).2,低噪声放大器加带通滤波器（

Q=100(a

VERY

good

filter)，

centered

at

10

kHz）any

signal

in

a

100

Hz

bandwidth

will

be

detected(10kHz/Q).The

noise

in

the

filter

pass

band

will

be

50

µV(5

nV/Hz

x

100Hz

x

1000)and

the

signal

will

still

be

10

µV.The

output

noise

ismuchgreater

than

the

signal,but

an

accurate

measurement

can

not

be

made.3,低噪声放大器加相敏检测器(PSD).

The

PSD

can

detect

the

signal

at10kHz

with

a

bandwidth

as

narrow

as

0.01

Hz!In

this

case,the

noise

in

thedetection

bandwidth

will

be

only

0.5

µV(5

nV/Hz

x