2. Circular Dichroism
Part I. Introduction

3. Circular Dichroism
Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. The absence of regular structure results in zero CD intensity, while an ordered structure results in a spectrum which can contain both positive and negative signals.
Jasco J-810 Circular Dichroism System

4. Chiral structure can be distinguished and characterized by polarized light
Optical rotation: the rotation of linearly polarized light by the sample
Optical rotary dispersion: the variation of optical rotation as a function of wavelength. The spectrum of optical rotation.
Circular Dichroism: the difference in absorption of left and right circularly light.

5. Types of polarized light
Plane polarized light consists two circularly polarized components of equal intensity
Two circularly polarized components are like left- and right-handed springs
As observed by looking at the source, right-handed circularly polarized light rotates clockwise
Frequency of rotation is related to the frequency of the light
Can be resolved into its two circularly polarized components
When added together after passing through an optically isotropic medium, plane polarized light results

7. Linear Polarized Light
C: speed of the light in vaccum; V: frequency; Lamda: wavelength

8. Circular Polarized Light
Passing plane polarized light through a birefringent plate (in the z-direction) which splits the light into two plane-polarized beams oscillating along different axes (e.g., fast along x and slow along y). When one of the beams is retarded by 90º (using a quarter-wave retarder) then the two beams which are now 90º out of phase are added together, the result is circularly polarized light of one direction. By inverting the two axes such that the alternate beam is retarded than circularly polarized light of the other direction is generated.
The result of adding the right and left circularly polarized that passes through the optically active sample is elliptically polarized light, thus circular dichroism is equivalent to ellipticity

9. Circularly Polarized Light
Left-handed
right-handed

10. Optical rotary dispersion
If the refractive indices of the sample for the left and right handed polarized light are different, when the components are recombined, the plane-polarized radiation will be rotated through an angle 
nl, nr are the indices of the refraction for left-handed and right-handed polarized light
 is in radians per unit length (from )

11. Optical Rotation n refractive index l wavelength of light
f angle of rotation

12. Optical Rotation
Usually reported as a specific rotation [], measured at a particular T, concentration and  (normally 589; the Na D line)
Molar rotation [] = []MW10-2

13. Optical rotary dispersion
Concentration of an optically active substance, c’, expressed in g cm-1 (as density of a pure substance)
d’ = thickness of the sample in decimeters
M = molecular weight of the optically active component
the 10-2 factor is subject to convention and is not always included in [M]

14. Optical rotary dispersion
M = molecular weight of the optically active component
n. b. the 10-2 factor is subject to convention and is not always included in [M]

15. Optical rotary dispersion
ORD curve is a plot of molar rotation [] or [M] vs 
Clockwise rotation is plotted positively; counterclockwise rotation is plotted negatively
ORD is based solely on the index of refraction
So-called plain curve is the ORD for a chiral compound that lacks a chromophore
Chiral compounds containing a chromophore can give anomalous, or Cotton effect, curves

16. Cotton Effect
Positive Cotton effect is where the peak is at a higher wavelength than the trough
Negative Cotton effect is the opposite
Optically pure enantiomers always display opposite Cotton effect ORD curves of identical magnitude
Zero crossover point between the peak and the trough closely corresponds to the normal UV max

17. Circular Polarized Light

18. Circular Polarized Light

19. Circular dichroism
Measurement of how an optically active compound absorbs right- and left-handed circularly polarized light
All optically active compounds ex-hibit CD in the region of the appropriate absorption band
CD is plotted as l-r vs 
For CD, the resulting transmitted radiation is not plane-polarized but elliptically polarized

20. Circular Dichroism q ellipticity l path length through the sample
A absorption

21. Circular dichroism
 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 []

22. Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase
different absorption of the left- and right hand polarized component leads to ellipticity (CD) and optical rotation (OR).

23. Circular Dichroism
The difference between the absorption of left and right handed circularly-polarised light and is measured as a function of wavelength. CD is measured as a quantity called mean residue ellipticity, whose units are degrees-cm2/dmol.

24. ORD and CD CD plots are Gaussian rather than S-shaped.
Positive or negative deflections depend on the sign of  or [] and corresponds to the sign of the Cotton effect
ORD spectra are dispersive (called a Cotton effect for a single band) whereas circular dichroism spectra are absorptive. The two phenomena are related by the so-called König-Kramers transforms.
Maximum of the CD occurs at the absorption max
Where more than one overlapping Cotton effect, the CD may be easier to interpret than the ORD with overlapping S-shaped bands

25. ORD spectra are dispersive (called a Cotton effect for a single band) whereas circular dichroism spectra are absorptive. The two phenomena are related by the so-called König-Kramers transforms.

28. Sample Preparation
Additives, buffers and stabilizing compounds: Any compound which absorbs in the region of interest ( 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.

29. 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.

30. 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.

31. 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

32. Part II. CD spectra of Nucleocide
Circular Dichroism
Part II. CD spectra of Nucleocide

33. Electron Molecular Energy

34. Chromophores of Nucleic Acid
p  p* transitions begin about 300 nm
n  p* buried under p  p* transitions
The intensity of the CD is low because it is a secondary effect of the asymmetric sugar inducing a CD in the chomophoric, but symmetric base. 

35. Chromophores of Nucleic Acid
Absorption Spectra
CD Spectra

36. Base Stacking & CD spectra of Nucleic Acid
The benzene-like p-electron systems of the bases make them hydrophobic, so the bases tend to stack in hydrogen bonding solvents to minimize the p-electron surface area exposed to the solvent.
The NH, NH2 and CO groups are hydrophilic, so the edges of the bases will interact well with hydrogen bonding solvents
For nucleic acids the hydrophobic planes, the hydrophilic edges and charge-charge interactions cause the bases to stack and the polymers to assume a helical structure.
The electronic transitions of the chromophoric bases are in close proximity, and can interact to give CD spectra of high intensity.

37. Polymorphic properties of Nucleic Acid
Polymorphism of nucleic acid in secondary structure
Number of base pairs per turn
Inclination of the base with respect to the helix axis
The distance of the bases from the helix axis
The rise per base-pair
Handedness of the helix
CD can measure the change in secondary structure as a function of solvent conditions.

38. From monomer to polymer
Formation of helical structure is a super asymmetry that gives rise to degenerate interactions between chromophoric bases and results in intense CD spectra
dimer
monomer
The conservative bands in the CD spectrum of a biopolymer that arise from the degenerate interaction among the electron transition dipoles of the chromophores attest to the presence of secondary structure.
CD of single stranded oilgo(rA) in aqueous solution at pH 7

39. Base-stacked helices in aqueous solution
Spectra is Composition dependent
CD of single stranded poly(rA)
CD of single stranded poly(rC)

40. CD of double-stranded DNA and RNA

41. CD vs. Absorption CD occurs only where normal absorption occurs
Native DNA
Denatured Native DNA
CD occurs only where normal absorption occurs
CD is more complicated revealing bands that not separated in the normal absorption spectrum
Average spectrum for the four component deoxynucleotides

42. Structure of DNA
A-DNA
B-DNA
Z-DNA

43. Discovery of Z-form DNA
Pohl and Jovin (1972 JMB, 67,p375 ) were the first to observe the
left-handed Z-form of poly(dGC)-poly(dGC), and they did this by using
circular dichroism spectroscopy.
The Z-form DNA
negative band at 290
positive band at 260 nm.
crossover about 185 nm
Z-form is not the mirror image of the B-form, the blue shift of the 200 nm of the B-form to 185 nm in the Z-form appears to be the trademark of the B to Z transition.
A-DNA
B-DNA
Z-DNA

44. DNA Secondary Structure & CD Spectra
A DNA
260 nm positive
210 nm intense negative
190 nm intense positive
A-DNA
B DNA
275 nm positive
240 nm negative
258 nm crossover
B-DNA

45. Sprecher et.al. Biopolymer 17,1009
The CD of E. coli DNA in various structure
A-DNA
10.4 B-DNA
10.2 B-DNA
Sprecher et.al. Biopolymer 17,1009

46. Solvent Effect on DNA Structure I
Calf thymus DNA
0% methanol
25% methanol
10.4 base pair B-form
50% methanol
65% methanol
75% methanol
95% methanol
10.2 base pair B-form

47. Solvent Effect on DNA Structure II
90% methanol
Titration with ethanol causes the same changes as with methanol in CD up to 65%. Adding more ethanol causes a change to A form
75% methanol
70% methanol
65% methanol

48. P Form DNA P-DNA (95% methanol/5% buffer) 330C
P-DNA (47.5% methanol/5% buffer/4.5% ethanol) 80C
The backbones are colored purple, and the bases are colored blue (guanine) and yellow (cytosine). The anionic oxygens of the phosphate groups are shown in red.
B-DNA
P-DNA
10.2 B-DNA (95% methanol/5% 80C)

49. Temperature Effect on DNA
The CD of poly(dA) poly(dT) as a function of temperature
44.70 C
38.80 C
CD is sensitive to the change in conformation when DNA melts with increasing temperature
10 C

50. Triplex Nucleic Acid 177 Triplex Poly(dA dT dT) 190 260 280 210 Duplex
250

51. With Mg+2
Intra-molecular triplex
Without Mg+2
With Mg+2
Intra-molecular triplex

52. Influence of the temperature on the parallel triplex
30C triplex
260nm
280nm
660C duplex
930C
denature

53. Part III. CD spectra of Protein
Circular Dichroism
Part III. CD spectra of Protein

54. Amide Chromphore n  p* centered around 220 nm
p  p* centered around 190 nm
n -> p* involves non-bonding electrons of O of the carbonyl;
p -> p* involves the p-electrons of the carbonyl

57. Random coil
positive at 212 nm (p->p*)
negative at 195 nm (n->p*)
b -Sheet
negative at 218 nm (p->p*)
positive at 196 nm (n->p*)
a-helix
positive (p->p*)perpendicular at 192 nm
negative (p->p*)parallel at 209 nm
negative at 222 nm is red shifted
(n->p*)

61. Secondary Structure Determination

68. TFE Induced Helix

69. Sodefrin&S10K-A3之CD測量圖

70. Helix Content = 100×(〔θ〕222／max〔θ〕222)
max〔θ〕222=-40,000[1－(2.5/n)]，
n=胺基酸之殘基數
Behrouz Forood et.al Proc.Natl.Acad.Sci.USA Vol.90,pp.838～842,February 1993

76. Applications of CD in Structural Biology
Determination of secondary structure of proteins that cannot be crystallised
Investigation of the effect of e.g. drug binding on protein secondary structure
Dynamic processes, e.g. protein folding
Studies of the effects of environment on protein structure
Secondary structure and super-secondary structure of membrane proteins
Study of ligand-induced conformational changes
Carbohydrate conformation
Investigations of protein-protein and protein-nucleic acid interactions

79. Software for the Analysis of Circular Dichroism Data
Tools for analyzing circular dichroism data :
LINCOMB and MLR( The method of least squares)
CONTIN   (The ridge regression procedure of Provencher and Glöckner)
VARSLC (The Variable Selection Method of Johnson and Coworkers )
SELCON (The Self-Consistent Method of Sreerama and Woody )
K2D.(A neural net analysis program of Andrade et al)
CCA (The convex constraint algorithm of Fasman and coworkers )
SVD (Singular Value Decomposition ).