Download Circular Dichroism?

Circular Dichroism
Circular Dichroism?
 원편광 이색성
 CD spectroscopy measures differences in the
absorption of left-handed polarized light versus
right-handed polarized light which arise due to
structural asymmetry
 분광학 (Spectroscopy)
 물질과 전자기파(빛)의 상호작용을 연구,
물질에서 방출되거나 물질에 흡수되는
스펙트럼을 분석하여 물질을 분석
Circular Dichroism?
 단백질 거울상(chiral) 이상질체인 D form과
L form으로 구성
 D/ L form이 동일한 양으로 구성된
화합물을 Racemic mixture
 편광된 빛이 asymmetric medium을
통과하면 optical rotation과 polarization
loss 일어남
 Ellipticity(loss)를 측정하여 ellipticity
크기와 경향을 diagram으로 나타낸 것이
Circular Dichroism
Light
 As an electromagnetic wave
 Electrical component
 Magnetic component
 Direction
 Two component는 서로 수직
 빛의 진행방향에도 서로 수직
Polarization(편광)
 진행방향에 대해 수직으로 진동
= 횡파의 특성
 횡파의 경우, 진동방향이 파동의
진행방향의 수직인 평면상에 놓여있게
되어 벡터의 성격을 갖게됨
 편광 = 파동의 진동이 그 평면상의 특정
방향으로 놓이게 되는 것
 편광방향 = 진동방향(물질에 더 큰 영향을
주는 전기장의 진동방향)
Polarization(편광)
 빛은 짧은 길이의 무수히 많은
파동줄기(wave train)가 모여서 형성된
것이므로 하나하나의 편광상태가 어떻게
집합되어 있는가를 고려해야 하므로
통계적인 처리가 필요
 빛은 물질과 반응하여 편광상태가 바뀔
수 있음
Polarization(편광)
 선형편광 = 편광방향이 바뀌지 않음
 전기장이 x 방향으로 진동하는 경우를 x
선형편광, y 방향 진동의 경우 y 선형편광
 x, y 선형편광을 적절히 조합하면 임의의
방향의 선형편광이나, 원형편광도 만들어
낼 수 있다.
Polarization(편광)
 전기장의 방향은 나선 형태로 계속 변함
 z 축 상의 한 점에 정지 한 채로 보면
다가오는 빛의 전기장은 점차 반
시계방향으로 회전을 하게 되는 것을 알
수 있다(좌향-원형편광)
Polarization
Dichroism
 The difference between the absorption
of left and right handed circularlypolarized light is measured as a
function of wavelength
Principle of CD
 Chiral molecules은 좌원편광, 우원편광
된 빛을 다르게 흡수 -> CD spectrum
 Chiral molecules은 optical active
 Optical activity = 편광 된 빛이 물질을
통과할 때 편광 면을 회전시키는 성질
(a)
(b)
 (a) Linear polarized light can be viewed as a
superposition of opposite circular polarized light of
equal amplitude and phase
 (b) Different absorption of the left- and right hand
polarized component leads to ellipticity (CD) and
optical rotation (OR)
  is therefore the angle between the initial plane of
polarization and the major axis of the ellipse of the
resultant transmitted light
 A quantity  is defined such that
tan  is the ratio of the major and minor axis of the
ellipse of the transmitted light
 ’ approximates the ellipticity
 When expressed in degrees, ’ can be converted to a
specific ellipticity [] or a molar ellipticity []
 CD is usually plotted as []
specific ellipticit y    

c' d
2




molar ellipticit y  θ  M  10
ε l  ε r  0.3032 103 θ
스펙트럼
 자외선 가시광선에 의해 분자나 원자가
에너지를 흡수하여 전이를 일으켜 발생
 흡수된 빛의 파장은 낮은 에너지준위의
전자를 높은 에너지 준위로 이동시킬 수
있는 에너지가 있음
전자전이
 결합분자 궤도 함수(bonding moleculer
orbital):  ,  (에너지 준위가 낮음)
 반 결합 분자궤도 함수(antibonding
moleculer orbital):  *, * (에너지 준위가
높음)
 비결합 전자 (nonbonding electron): n 전자
(유기 화합물 중 O, N, S 및 할로겐 원자)
 전자의 전이는 전자가 결합성 궤도
함수(bonding orbital)에서 반결합성 궤도
함수(anti-bonding orbital)로 옮아가는 것을
의미함
Application
 Near UV CD (250 - 350 nm)
 Near-UV CD spectroscopy is dominated by
Phe, Tyr, Trp and disulfides
 When an aromatic residue is held rigidly in
space, its environment is asymmetric, and it
will exhibit CD
Application
 Far UV CD (180 - 250 nm)
 The amide group is the most abundant
CD chromophore in proteins
 In secondary structure conformations,
the backbone and the amide bond
chromophores are arranged in regular,
organized, asymmetric patterns
Application
 n -> π* centered around 220 nm
 n -> π* involves non-bonding
electrons of O of the carbonyl
 π -> π* centered around 190 nm
 π -> π* involves the π-electrons of
the carbonyl
 The intensity and energy of these
transitions depends on φ and ψ
(i.e., secondary structure)
Application
 Far UV-CD of random coil:
 positive at 212 nm (π->π*)
 negative at 195 nm (n->π*)
 Far UV-CD of β-sheet:
 negative at 218 nm (π->π*)
 positive at 196 nm (n->π*)
 Far UV-CD of α-helix:
 Exciton coupling of the π->π* transitions leads
to positive (π->π*)(perpendicular) at 192 nm
and negative (π->π*)(parallel) at 209 nm
 negative at 222 nm is red shifted (n->π*)
Application
 Determination of secondary structure of
proteins that cannot be crystallized
 Investigation of the effect of e.g. drug
binding on protein secondary structure
 Studies of the effects of environment on
protein structure
 Study of ligand-induced conformational
changes
Application
 광우병 진단
 정상적인 cellular prion protein 의 경우,
단백질의 2차 구조가 대부분 alpha-helix로
이루어져 있다.
 반면, 광우병에 걸린 scrapie prion protein은
beta structure 가 대부분의 2차 구조 구성
성분으로 변하게 된다.
 Alpha helical structure 에서 beta-structure
로의 변화를 CD를 통해 확인할 수 있고,
광우병의 발병유무를 측정가능하다.
Sample Preparation
 Additives, buffers and stabilizing compounds:
Any compound which absorbs in the region of
interest (250 - 190 nm) should be avoided.
 A buffer or detergent or other chemical should
not be used unless it can be shown that the
compound in question will not mask the
protein signal.
Sample Preparation
 Protein solution: From the above follows
that the protein solution should contain only
those chemicals necessary to maintain
protein stability, and at the lowest
concentrations possible. Avoid any
chemical that is unnecessary for protein
stability/solubility. The protein itself should
be as pure as possible, any additional
protein or peptide will contribute to the CD
signal.
Sample Preparation
 Contaminants: Unfolded protein, peptides,
particulate matter (scattering particles),
anything that adds significant noise (or
artifical signal contributions) to the CD
spectrum must be avoided. Filtering of the
solutions (0.02 um syringe filters) may
improve signal to noise ratio.
 Data collection: Initial experiments are
useful to establish the best conditions for
the "real" experiment. Cells of 0.5 mm path
length offer a good starting point.
Sample Preparation
 Nitrogen purging: the function of
purging the CD instrument with nitrogen
is to remove oxygen from the lamp
housing, monochromater, and the
sample chamber. The reason for
removing oxygen is that oxygen absorbs
deep UV light, thus reducing the light
available for the measurement.
Sample Preparation
 Typical Initial Concentrations:
 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
 Need very little sample: 0.1mg
Advantage




Simple and guick experiment
No extensive preparation
Measurements on solution phase
Relatively low concentrations/amounts of
sample
 Microsecond time resolution
 Any size of macromolecule
Disadvantage
 Difficult to quantitate similarity or
differences
 Certain buffer components absorb strongly
in Far-UV & can cause interference
Conclusion
 편광된 빛이 asymmetric medium을
통과하면 optical rotation과 polarization
loss 일어남
 Ellipticity(loss)를 측정하여 ellipticity
크기와 경향을 diagram으로 나타낸 것이
Circular Dichroism
 Near UV CD (250 - 350 nm)
 Near-UV CD spectroscopy is dominated by
Phe, Tyr, Trp and disulfides
Conclusion
 Far UV CD (180 - 250 nm)
 In secondary structure conformations,
the backbone and the amide bond
chromophores are arranged in regular,
organized, asymmetric patterns
 Random coil, β-sheet, α-helix