Circular Dichroism

Basics
Dichroism is the phenomenon in which light absorption differs for
different directions of polarization.
 Light

EM-wave

Polarization of light
Polarization
 Vertically Polarized Light

Vertically (y axis) polarized wave having an amplitude A, a wavelength of λ, and an

angular velocity (frequency * 2π) of ω, propagating along the x axis.
 Plane-Polarized Light

Vertical

Horizontal
Vertically polarized Light - The electric vector vibrations occur in the vertical plane.
Light emitted from a source is normally not polarized at all, and the axis of the electric
vector can take all angles to the normal.
 Plane Polarized Light Generation
Plane polarized light can be generated using
1. Polaroid,
2. Nicol prism
3. Glan-Thomson Prism

http://www.microscopyu.com/tutorials/java/polarized/calcite/index.html
Generating Circularly Polarized Light

Circularly polarized light is generated by 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 (x and 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.

The two axes are inverted to generate circularly polarized light of the other direction.

The result of adding the right and left circularly polarized that passes through the optically active
sample is elliptically polarized light
 Circularly Polarized Light Generation
Circularly polarized light can be generated using
1. Pockel Cell
2. Photoelastic Modulator (or Electro-optic modulator)

http://www.microscopyu.com/tutorials/java/polarized/calcite/index.html
 Circularly Polarized Light

Right Circular

Left Circular
 Interaction of Light and Matter: Absorption

Material with an
extinction coefficient ε

The light gets weaker (its amplitude

drops)

In Out
 Interaction of Light and Matter: Refraction

Material with an index of

refraction n

The light slows down inside the

material, therefore its wavelength
becomes shorter and its phase gets
shifted

In Out
 Circular Birefringence

Material having different

refraction indices for
right and left circularly
polarized lights: nR and
nL

The plane of polarization of plane-

polarized light gets rotated

In Out
 Circular Dichroism

Material having different

extinction coefficients
for right and left
circularly polarized
lights: εR and εL

Plane-polarized light becomes

elliptically polar

In Out
 Circular Dichroism And Birefringence

Material having different

extincion coefficients
AND refraction indices
for right and left
circularly polarized
lights: εR and εL AND nR
and nL

Plane polarized light gets elliptically

polar, with the great axis of the
ellipse being rotated relative to the
original plane of polarization
In Out
Optical Rotatory Dispersion (ORD)
 Specific Rotation

Enantiomers (optical isomers, non superimposable mirror images) possess the

property of allowing the rotation of plane polarized light.

Optical activity is a unique property characteristic of enantiomers.

It is measured by means of a Polarimeter

Basic Polarimeter Setup

Polarimeter is the instrument used to measure the angle of rotation of a
plane polarized light. The magnitude of rotation depends on:
i) Nature of the compound
ii) Concentration of the solution
iii) Thickness of the layer traversed by polarized light
iv) Wavelength of the light used
v) Temperature of the solution.
 Optical Rotatory Dispersion (ORD)

ORD – Optical rotatory dispersion (ORD) spectroscopy is a technique

for measuring the ability to rotate the plane of polarization, as a
function of wavelength.

The technique of polarimetry essentially measures the angle through

which the plane of polarization is changed after such light is passed
through a solution containing a chiral (optically active) substance.

The extent and even the sign of optical rotation differs at different
wavelengths. The wavelength dependence of the optical rotation is
known as optical rotatory dispersion.
 Optical Rotary Dispersion

ORD curve is a plot of molar rotation [α

α] or [M] vs
λ

Clockwise rotation is plotted positively;
counterclockwise rotation is plotted negatively

ORD is based solely on the index of refraction

Plain curve is the ORD for a chiral compound
that lacks a chromophore
 Circular Dichroism (CD)
The technique of ORD has been replaced by circular dichroism (CD)
spectroscopy, which gives rather better information about the three
dimensional structure of macromolecules containing chiral centres.

Circular dichroism (CD) spectroscopy measures the difference 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.

In CD, circularly polarized light is used and this is obtained by superimposing

two plane polarized light waves of the same wavelengths and amplitudes but
differing in phase by one quarter of a wavelength and in their planes of
polarization by 900

CD spectrum is a plot of ellipticity versus wavelength.

In polarimetry the specific rotation [α α]λ would be measured, whereas in CD
spectroscopy it is the ellipticity, θ, which is measured.

Circular Dichroism is the difference in absorption between left and right hand
circularly polarized light in chiral molecules.
∆ε = εl - εr
Example of circular dichroism in glucose, a simple sugar.
 Physical Principles of CD

Chiral or asymmetric molecules produce a CD spectrum

because they absorb left and right handed polarized light
to different extents and thus are considered to be
"optically active"
 Circular Dichroism

The difference between the absorption of left and right

handed circularly-polarized 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.
The difference in molar extinction coefficients is given
by
εL-εR =∆ε (liter.mol-1·cm-1).
The molar ellipticity [θ
θ] is related to the difference in
extinction coefficients by
[θ
θ] = 3298 ∆ε.

Ellipticity
Plane polarized light is the sum of left and right circularly polarized light. When
plane polarized light passes through a solution containing an optically active
substance the left and right circularly polarized components of the plane-
polarized light are absorbed by different amounts. When these components are
recombined they appear as elliptically polarized light.
Defn : The ellipticity is defined as Q, the angle of polarization and is measured
in degrees, (deg) or millidegrees, (mdeg).
Ellipticity, θ
 Circular Dichroism

In proteins, CD of the amide chromophore reports on secondary structure

(proportions of alpha helix, beta sheet, turns etc)
 ORD and CD

CD plots are Gaussian rather than S-shaped.

Positive or negative deflections depend on the

sign of ∆ε or [θ] and corresponds to the sign of the
Cotton effect

Maximum of the CD occurs at the absorption λmax

Where more than one overlapping Cotton effect,

the CD may be easier to interpret than the ORD
with overlapping S-shaped bands
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
Krönig-Kramers transforms.
CD, ORD and Absorbance spectra of R and S forms of
camphor sulphonic acid.
Applications of CD

 Secondary structure determination

Determination of secondary structure of proteins that cannot be
crystallized. Advantage when X-ray/Nmr structure cannot be
obtained
e.g. Secondary structure of membrane proteins

 Study of conformation of biological macromolecules and

complements data generated by NMR – Major application

 Study of conformational changes during, or because of interaction

with other entities
e.g.
• determination of binding constants of substrates, cofactors,
inhibitors or activators of virtually any enzyme.
• Investigation of the effect of interaction (e.g. drug binding) on
protein secondary structure
• Study of active site changes
 Gives relative proportions of α–helical, β-sheet and random coil
in solution

 Study dynamic processes e.g. protein folding

 Studies of the effects of crystallization media on protein

structure (Useful for X-ray people)

 Application of CD to tertiary structure is limited, owing to

inadequate theoretical understanding of the influences of different
parts of the molecules at this level of structure.

In short, CD can be used for

• Protein/Peptide conformational studies
• Thermal and chemical denaturation studies
• Structure determination
 Secondary Structure from CD Spectra

CD is particularly
useful for measuring
the temperature
dependence of
protein secondary
structure.
 Components of a CD spectrometer

→ →
Prism Polarizer
 The CD Signal

For biological samples, IAC is typically 104 times smaller than IDC
CD Instrumentation
 Ideal Light Source for CD

High flux

Stable linear polarization

Extended wavelength range (VUV – Near IR)

Wavelength range 180 – 600 nm

180 – 800 nm (modern instruments)
Practical Considerations
Solvent Cut-Off (A=1.0) for Two Different Cell Pathlengths

Compound 1.0 mm 0.05 mm

H2O 182 176

F6iPrOH 174.5 163

F3EtOH 179.5 170

MeOH 195.5 184

EtOH 196 186

MeCN 185 175

Dioxane 231 202.5

Cyclohexane 180 175

n-Pentane 172 168

 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.

Protein solution: 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.

Contaminants: Unfolded protein, peptides, particulate matter
(scattering particles), anything that adds significant noise (or
artificial 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.
 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
Applications
Circular dichroism spectroscopy is particularly good for:

• determining whether a protein is folded, and if so characterizing its

secondary structure and tertiary structure

• comparing the structures of a protein obtained from different sources

(e.g. species or expression systems) or comparing structures for
different mutants of the same protein

• demonstrating comparability of solution conformation and/or thermal

stability after changes in manufacturing processes or formulation
[comparability protocols]

• studying the conformational stability of a protein under stress --

thermal stability, pH stability, and stability to denaturants -- and how
this stability is altered by buffer composition or addition of stabilizers
and excipients.
• CD is excellent for finding solvent conditions that increase the
melting temperature and/or the reversibility of thermal unfolding,
conditions which generally enhance shelf life

• determining whether protein-protein or protein-ligand interactions

alter the conformation of protein.

• If there are any conformational changes, this will result in a spectrum

which will differ from the sum of the individual components. Small
conformational changes have been seen, for example, upon formation
of several different receptor/ligand complexes.
Far-UV CD is used for determining protein structure, amide bond electrons absorb in this
energy range:

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

The intensity and energy of these transitions depends on the angles the peptide bond
assumes (Φ,Ψ angles) and therefore on the secondary structure of the protein.
 Examples of different pure secondary structures

In a folded protein the amide is in a continuous array. For example, the absorption
spectrum of poly-L-lysine in an α-helix, β-sheet, and unordered (random coil) differ due
to long-range order in the amide chromophore:
 Far-UV CD spectrum thermal denaturation of lysozyme

The above figure illustrates clearly the change in CD signature as a function of

temperature. Comparison of known CD signatures with the data in this experiment
suggests that the initial CD signature is a combination of α-helical and β-sheet
structure which, on heating, loses much of it’s α-helical content to an unfolded state,
whilst retaining some β-sheet character.

http://www.photophysics.com/prot_struct.php
Dependence of chain length on CD spectrum
Generation of basis-set CD spectra

Circular dichroism spectra of "pure" secondary structures. Use far-UV

CD to determine amounts of secondary structure in proteins by
generate basis sets by determining spectra of pure a-helix, b-sheet,
etc. of synthetic peptides or deconvoluting CD spectra of proteins
with know structures to generate basis sets of each of secondary
structure poly-L-lysine can adopt 3 different conformations merely by
varying the pH and temperature:
• random coil at pH 7.0
• a-helical at pH 10.8
• b-sheet form at pH 11.1 after heating to 52°C and recooling.
Abs Spt and CD Spectra of Poly-Lysine

UV-spectrum Far UV-CD spectrum

CD of Subtilisin

Image of subtilisin showing its numerous helical, beta sheet and looped regions.

Source : http://www.ruppweb.org/cd/subtilis.htm
 Secondary carboxypepti α-
Myoglobin
Technique Structure dase chymotrypsin
α 23% 8% 68%
X-ray
crystallograph β 18% 22% 0%
y
RC + other 59% 70% 32%

α 13% 12% 68%

CD using
(Lys)n Basis β 31% 23% 5%
Sets
RC + other 56% 65% 27%
 Aromatic amino
Protein backbone.
acids.
CD sensitive to
CD sensitive to
secondary
tertiary structure
structure change.
change.

170 nm Far-UV 250 nm NearUV 320 nm

Structural types Structural types

identified: identified:
Alpha helix Phenylalanine
Beta sheet Tyrosine
Beta turn Tryptophan
Polyproline Disulphide bonds
Irregular

Relationship between regions of the CD spectrum and protein structural types.

Changes in the circular dichroism spectra of bio-molecules represent changes in the
structure of the protein
 CD for a Dinucleotide

Spectral differences between monomeric residues

nucleic acid bases themselves are directly involved in close interactions in all common
secondary structures

E.g CD spectrum of the dinucleoside phosphate ApG is different from the sum of the
CD spectra of A and G monomers. The CD spectra of ApG and GpU are the average
of the two monomers plus and an additional term to account for the base-base
interactions.

In a trinucleoside diphosphate such as ApGpU, there would be contributions from

three monomers, two interactions between neighboring bases, and a final contribution
due to interaction between the next-nearest neighbors, A and U.
Nucleic acids CD spectra complicated
 Studies on Nucleic Acids

Melting studies can also be conducted by CD.

Changes in conformation can be monitored as a

function of temperature and solvent.
 First Observation of Z-form DNA by CD

Pohl and Jovin (1972) were the first to observe the left-handed Z-
form of poly(dGC).poly(dGC) using CD.

Z-DNA left handed

Pohl, F.M., and T.M.Jovin (1972) J. Mol. Biol. 67, 375-396

Summary Of Strengths And Weaknesses Of CD

Strengths
Uses very little sample (200ul of 0.5 mg/ml solution in standard cells), non-
destructive.
Relative changes due to influence of environment on sample (pH, denaturants,
temperature etc.) can be monitored very accurately.
CD is rapid unlike NMR, X-ray
CD has an important role in the structure determination of proteins
Analysis of structural changes in a protein upon some perturbation
Comparison of the structure of an engineered protein to the parent protein

Weakness
CD cannot give accurate structural details like NMR, X-ray
interference with solvent absorption in the UV region, only very dilute, non-
absorbing buffers allow measurements below 200 nm.

Absolute measurements subject to a number of experimental errors, average accuracy

of fits about +/- 10%.

A CD spectropolarimeter is relatively expensive.

Conclusion
CD is rapid and can be used to analyze a number of candidate proteins from
which interesting candidates can be selected for more detailed structural analysis like
Advanced Topics
 Vibrational Circular Dichroism

All the above discussion centered around

electronic CD.

CD can be measured for any type of transition,

even vibrational band.

Vibrational CD (VCD) spectra have the

advantage over electronic CD in that the bands
are well separated and correspond to specific
functional groups.
References
• Biophysical Chemsitry, Nath & Upadhyay.Himalaya Publishing House (2005).

• Principles of Physical Biochemistry, K.E.Van Holde, W. C. Johnson and P.S. Ho,

II Edition, Pearson Education International

• Biophysical Chemistry, Part II: Techniques for the study of Biological Structure
and Function, C.R.Cantor and P.R.Schimmel

• Principles of Analytical Techniques, Wilson & Walker; Cambridge; 5th edition,

(2003).

• Principles of Instrumental Analysis , A.Skoog, F. J. Holler, T. A. Nieman ;Harcourt

Brace College Publishers , 5th edition,(1998)