同步輻射圓二色光譜技術(shù)

同步輻射圓二色光譜技術(shù)中國(guó)科技大學(xué)國(guó)家同步輻射2008-041同步輻射圓二色光譜技術(shù)1，圓二色光譜概念及其意義2，基本原理3，同步輻射圓二色光譜的優(yōu)勢(shì)4，同步輻射圓二色光譜測(cè)量方法5，國(guó)家同步輻射

圓二色光譜儀器簡(jiǎn)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)物質(zhì)吸收?qǐng)A偏振光時(shí)，如

果對(duì)左旋和右旋圓偏振光

的吸收表現(xiàn)出一定的差異，則稱該物質(zhì)具有圓二色性。1516圓二色光譜的意義生物大分子如蛋白質(zhì)和DNA是由光學(xué)活性基團(tuán)組成，并且它們可以采取不同的三維結(jié)構(gòu)，因此，每一種分子都有特征的CD譜。研究蛋白質(zhì)和DNA最有用的光譜區(qū)是紫外和真空紫外區(qū)（160

to

300

nm)，因?yàn)閬碜缘鞍踪|(zhì)的縮氨酸主鏈和側(cè)鏈以及DNA的嘌呤和嘧啶的電子躍遷就發(fā)生在這一光譜能量區(qū)。氨基酸是蛋白質(zhì)的單體。天然存在于蛋白質(zhì)中的氨基酸共有20種。氨基酸由肽鍵相連形成多肽鏈。蛋白質(zhì)結(jié)構(gòu)17蛋白質(zhì)結(jié)構(gòu)的層次一級(jí)結(jié)構(gòu)氨基酸序列二級(jí)結(jié)構(gòu)主要由氫鍵穩(wěn)固的局部構(gòu)象，如-helix,-sheet等三級(jí)結(jié)構(gòu)三維構(gòu)象四級(jí)結(jié)構(gòu)多個(gè)多肽鏈的組合18蛋白質(zhì)的結(jié)構(gòu)CD19XRDNMR二級(jí)結(jié)構(gòu)與圓二色譜20二級(jí)結(jié)構(gòu)與圓二色譜-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[

]

,圓二色光譜的解析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圓二色光譜的解析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解譜程序25圓二色光譜的優(yōu)點(diǎn)簡(jiǎn)單、快捷樣品不需要做過多準(zhǔn)備在溶液狀態(tài)下測(cè)量相對(duì)少的樣品用量、低濃度微秒時(shí)間分辨適用于任何尺寸的大分子樣品26CD

vs

XRD（NMR)CD約30分鐘XRD、NMR幾天——幾個(gè)月：篩選、優(yōu)化結(jié)晶條件，得到蛋白質(zhì)晶體獲得衍射圖、確定相位建立模型并優(yōu)化，最終獲得結(jié)構(gòu)信息27測(cè)定不能結(jié)晶的蛋白質(zhì)的二級(jí)結(jié)構(gòu)研究結(jié)合在蛋白質(zhì)二級(jí)結(jié)構(gòu)上的藥物的功能動(dòng)態(tài)過程如蛋白質(zhì)折疊的研究研究環(huán)境對(duì)蛋白質(zhì)結(jié)構(gòu)的影響研究膜蛋白的二級(jí)及超二級(jí)結(jié)構(gòu)研究基質(zhì)引起的構(gòu)型變化碳水化合物構(gòu)型研究研究蛋白質(zhì)-蛋白質(zhì)及蛋白質(zhì)-核酸的相互作用識(shí)別折疊狀態(tài)圓二色光譜的意義基本原理——數(shù)學(xué)描述

AClI

I010

I010A

AL

AR

LCl

RCl

028圓二色定量描述：對(duì)左旋和右旋圓偏振光吸收度的差異y’y

L

=

tg-1[(|ER

-

EL|)/(ER

+

EL)]圓二色性的描述——橢圓率θElErxRx’基本原理——數(shù)學(xué)描述29旋光色散與圓二色性1

光在介質(zhì)中的Rate:

c

/

n

,

(n

折射率) n

=fdirection光強(qiáng)I:A=lg(I0

/I)=Cl

f2

ORD(optical

rotatory

dispersion)?圓雙折射(Circular

birefringence):nl導(dǎo)致左、右旋圓偏振光相位不同nrChiral

compd.Optical

active

object

L

nn

=

nl

-

nr

f

(

)偏轉(zhuǎn)角度(旋光度)與波長(zhǎng)所建立起的函數(shù)關(guān)系3031比旋光度

[]

(the

specific

rotation)摩爾比旋光度

[]

(the

molar

rotation)平均殘基旋光度

[m]

(the

mean

residue

rotation)[]

=

/Cl[]

[]

[Mw/100][m]

=

[]

[MRw/100]單位:(*****)C

:

g/mLl

:

dmMw:

molecular

weightMRw:

the

mean

residue

Mw旋光色散與圓二色性旋光色散與圓二色性3

CD

(circular

dichroism)光強(qiáng)不同、或左右旋圓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比橢圓率)[

]

Cw

l[

]

Mw

[

]/100

(the

molar

ellipticity摩爾橢圓率)[

]MRw

MRw

[

]/100

(the

mean

residue

ellipticity平均殘基橢圓率)旋光色散與圓二色性[

]

100

Cl

=

3300

C:摩爾濃度

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——數(shù)學(xué)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——數(shù)學(xué)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常規(guī)圓二色光譜儀的組成鎖相放大器光彈調(diào)制器器36同步輻射優(yōu)越性同步輻射優(yōu)越性同步輻射優(yōu)越性真空紫外常規(guī)連續(xù)譜光源3940同步輻射優(yōu)越性光

源波長(zhǎng)范圍（nm）光譜輻射峰值功率（mW/mm2.sr.nm）氫燈（1KW）165——2500.1(在230nm)氙燈150——2250.01(在170nm)氪燈125——1650.001(在150nm)同步輻射X-ray——IR1同步輻射優(yōu)越性41同步輻射園二色光譜的優(yōu)越性42同步輻射園二色光譜的優(yōu)越性同步輻射園二色光譜的優(yōu)越性44同步輻射優(yōu)越性彎鐵彎鐵產(chǎn)生的同步輻射光的偏振特性：在 軌道平面內(nèi)是線偏振光，在軌道平面上方及下方是手性相反的橢圓偏振光45同步輻射優(yōu)越性水平與垂直偏振分量的歸一化強(qiáng)度與波長(zhǎng)、觀察角的關(guān)系46在這個(gè)波長(zhǎng)范圍，由于有幾種透光晶體材料，獲得圓偏振光的方法有些類似常規(guī)的做法，即首先獲得線偏振光，然后再通過光彈調(diào)制器得到圓偏振光。所不同的是，不再需要晶體線偏振器來獲得線偏振光，而是直接利用同步輻射光的偏振特性。紫外-真空紫外區(qū)（120nm≤λ≤300nm）同步輻射圓偏振光的獲得彎鐵利用偏振擋光板獲得線偏振同步輻射光，然后由光彈調(diào)制器轉(zhuǎn)換為圓偏振光（用于

國(guó)家同步輻射光源NSLS）球面光柵球面反射鏡狹

縫偏振擋光板光彈調(diào)制器47同步輻射圓偏振光的獲得彎鐵狹

縫光彈調(diào)制器利用鍍金反射鏡獲得線偏振同步輻射光，然后由光彈調(diào)制器轉(zhuǎn)換為圓偏振光鍍金反射鏡光柵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-resolution1004?997?1000?Spot

diagram

on

slit（600g/mm、1000?）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鎖相放大器及其工作原理1962年第一臺(tái)儀器問世，發(fā)現(xiàn)俄歇效應(yīng)。據(jù)統(tǒng)計(jì)，已在幾百種場(chǎng)合中得到應(yīng)用。

在弱信號(hào)探測(cè)儀器中鎖定放大器是一個(gè)

非常重要的品種。信噪比可低達(dá)10-5。BW=0.0004Hz（相當(dāng)于Q值=108）。兩個(gè)隨機(jī)過程的相關(guān)性對(duì)兩個(gè)信號(hào)x(t)和y(t)，定義相關(guān)函數(shù)–

互相關(guān)函數(shù)Rxy

,Ryx

：T

y(t

)

x(t

)dtT

/

2T

/

21yxR

(

)

TLim69T

T

/2T

/2x(t

)

y(t

)dt1TRxy

(

)Lim隨機(jī)過程的相關(guān)性–自相關(guān)函數(shù)：70T

)dtR(

)

T

/

2T

/

2x(t)x(t1TLim白噪聲，自相關(guān)函數(shù)=0自相關(guān)檢測(cè)積分器乘法器延遲S)Rss(τ)參考通道信號(hào)通道71自相關(guān)檢測(cè)設(shè)有信號(hào)S(t)，通過如上圖所示的功能器件，則：

)

(

ST

T

/21

T

/2R(

)lim

[T(

)

(

)][

(

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

T

/21

T

/2limTRSS

(

)RnS

(

)RSn

(

)Rnn

(

)RSS

(

)Rnn

(

)假定噪聲的平均值等于零7273正弦波與噪聲之和的自相關(guān)函數(shù)SignalS(t)V

cos(wt)21nn

R

(

)

)

VcosCorrelated function

R(積分器乘法器S)Rss(τ)參考通道信號(hào)通道延遲互相關(guān)檢測(cè)74互相關(guān)檢測(cè)設(shè)有信號(hào)S(t)，通過如上圖所示的功能器件，則：tS

StT

T

2/T

2/21())(][1R

)(

limT21

2S

SR

(

)RnS

(

)2((t)S(ST

T

/2T

/2211limT1

2S

SR

(

)75內(nèi)□前置被測(cè)信號(hào)濾波器主放靈敏度乘法器T積分器DC放大輸出參考信號(hào)產(chǎn)生振蕩相位乘法器的工作過程V+-接差動(dòng)積分器信號(hào)地參考信號(hào)77被測(cè)信號(hào)積分器工作過程低通濾波器及特性>f78100%乘法器（Phase-Sensitive

detector）輸出Vpsd

=

Vsigsin(rt

+

sig)

VLsin(Lt

+

ref)=

1/2

VsigVLcos([r

-

L]t

+

sig

-

ref)-1/2

VsigVLcos([r

+

L]t

+

sig

+

ref)當(dāng)r=L低通濾波器Vpsd

=

1/2

VsigVLcos(sig

-

ref)7980信號(hào)：10nV

sinewave

at

10

kHz1,

低噪聲放大器：

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,低噪聲放大器加帶通濾波器（

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,低噪聲放大器加相敏檢測(cè)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