CIRCULAR DICHROISM

Shantan ReddyVIT University

What is it?

• Circular dichroism is the difference in the absorption of left-handed circularly polarised light (L-CPL) and right-handed circularly polarised light (R-CPL).

• Occurs when a molecule contains one or more chiral chromophores.

Circular dichroism = ΔA(λ) = A(λ)LCPL - A(λ)RCPL (where λ is the wavelength)

Basics of Polarization

Linearly polarised light Circularly

polarised light

Linearly Polarised Light

In a linearly polarised light oscillations are confined to a single plane. All polarised light

states can be described as a sum of two linearly polarised states at right angles to each other –

vertically and horizontally polarised light.

Vertically polarised light

Horizontally polarised light

Superposition of the plane polarised waves

1. Linearly Polarised light

Horizontally and vertically polarised light waves of equal amplitude that are in phase with each

other will produce a resultant light wave which is linearly polarised at 45˚ and the properties of the resulting electromagnetic wave depends on the

intensities and phase difference of the component.

Linearly polarised light

• When one of the polarised states is out of phase with the other by a quarter-wave, the resultant will be a helix and is known as circularly polarised light (CPL).

• The optical element that converts between linearly polarised light and circularly polarised light is termed a quarter-wave plate. A quarter-will convert linearly polarised light into circularly polarised light by slowing one of the linear components of the beam with respect to the other so that they are one quarter-wave out of phase. This will produce a beam of either left- or right-CPL.

Circularly polarised light

2. Left Circularly Polarised light

3. Right Circularly Polarised light

Superposition of circularly polarised waves

The superposition of a left circularly polarized wave and a right circularly polarized wave of equal amplitudes and wavelengths is a plane

polarised wave.

Circular Dichroism

• Some materials possess a special property: they absorb left circularly polarized light to a different extent than right circularly polarized light. This phenomenon is called circular dichroism.

• Assume that a plane-polarized light wave (blue) traverses a medium that does not absorb the left circularly polarized component (red) of the wave at all but highly absorbs the right circularly polarized component (green).

The intensity of the green component decreases in comparison to the red one. The superposition of the two components yields a resulting field vector that rotates along an ellipsoid path and is called an elliptically polarized light.

• The direction of rotation of the elliptically polarized light is determined by the circular component that remains stronger after traversing the material.

• Real materials usually absorb both components, to a different extent.

• How elliptical the plane-polarized wave becomes after traversing the medium is determined by the difference between the absorptions of the two circularly polarized components.

Circular Birefringence

• There are materials having another special property: their refraction index is different for left and right circularly polarized light. This phenomenon is called circular birefringence.

• Assume that a plane-polarized light wave (blue) traverses a medium that does not slow down the left circularly polarized component (red) of the wave at all but slows down the right circularly polarized component (green) somewhat. For the latter component, the refraction index of the material is n=1.05.

This slowdown and the decreased wavelength is hard to see in the figure because the refraction index (1.05) is close to 1.0.

• Before the medium, the field vectors of the components coincide when they point vertically up or down. But after the light exits the medium, the superposition of the two circularly polarized components is a plane-polarized wave with a plane of polarization that is rotated by 36° with respect to the original polarization plane.

• Real materials usually have refraction indices greater than 1.0 (but not equal) for both circular components.

• The angle by which the polarization plane of the light exiting the medium rotates with respect to the original polarization plane is determined by the difference between the refraction indices for the two circularly polarized components.

Circular Dichroism & Circular Birefringence

The red component traverses the medium unchanged, but the medium has absorption and a refraction index with respect to the green component.

• The exiting light is no longer plane-polarized, it is not the plane of polarization that gets rotated but the big axis of the ellipse of polarization of the elliptically polarized light.

• With the appropriate instrument, the ellipticity and the angle of rotation of the polarization plane of light can be measured. From those data, the difference between absorptions and refraction indices with respect to left and right circularly polarized lights can be calculated.

• Circular dichroism and circular birefringence are caused by the asymmetry of the molecular structure of matter. The optical activity of solutions of biological macromolecules provides information about the structural properties of the macromolecules.

Physics of CD spectroscopy

• In an optically active sample with a different absorbance ‘A’, for the two components the amplitude of the stronger absorbed component will be smaller than the less absorbed component.

• A projection of the resulting amplitude yields an ellipse.

• Rotation of the polarization plane by a small angle ‘a’ occurs when the phases for the two circular components becomes different which requires a difference in the refractive index n by the effect called circular birefringence.

• The change of optical rotation with wavelength is called optical rotatory dispersion.

• CD and Optical rotation exist together and they are related by Kronig-Krames transformation.

• The difference between left and right handed absorbance is very small ( in the range of 0.0001)

• CD is a function of wavelength making it a characteristic of molecules.

Data Analysis

• The differential absorption is few 1/100ths to a few 1/10ths of a percent, but can be determined accurately.

• Raw data represents the ellipticity of the sample in millidegrees.

x (degrees)

• To be able to compare these ellipticity values they have to be converted into a normalized value, the mean molar ellipticity per residue.

• We need to consider path length ‘l’; concentration ‘c’ ; molar mass ‘M’ ; number of residues ‘nr‘.

Instrumentation

Prism

Nitrogen Purging?

• Removes oxygen from the lamp housing, monochromator and the sample chamber.

• The presence of oxygen is detrimental for 2 reasons – i) when deep UV light strike oxygen, ozone is produced, which degrades optics.

ii) oxygen absorbs deep UV light which reduces the amount available for measurement.

Sample Preparation and Measurements

• Additives, Buffers and Stabilizing compounds - any compound that absorbs in the region of interest (250 -190 nm) should be avoided.

• Protein solution – It should contain only those chemicals required to maintain protein stability at the lowest conc possible. Any additional protein or peptide will contribute to the CD signal.

• Data collection – Initial experiments are needed to establish the parameters for the actual experiment.

Typical Initial Conditions

• Protein Concentration – 0.5mg/ml• Cell path length – 0.5 mm• Stabilizers (metal ions, etc.) – minimum• Buffer concentration – 5mM or as low as

possible while maintaining protein stability.

Sample concentration effects

• Optimum absorbance to use is 0.89nm• For a 1mm path length cell, this absorbance is

achieved with a protein concentration of 0.1-0.3 mg/ml.

CD of proteins and polypeptides

For proteins we will be mainly dealing with the absorption in the UV region of the spectrum

originated from such chromophores as peptide bond, amino acid side chains (aromatic side chains of Phe, Tyr, and Trp have absorption

bands in the vicinity of 250-320 nm; the disulfide group is an inherently asymmetric chromophore that can lead to a broad CD absorption around

250 nm), and any prosthetic groups.

CD of Proteins – Far UV region

n -> π* centered around 220 nmπ -> π* centered around 190 nmn -> π* involves non-bondingelectrons of O of the carbonylπ -> π* involves the π-electrons of the carbonylThe intensity and energy ofthese transitions depends on φand ψ (i.e., secondary structure)

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:exiton 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*)

Far UV CD spectra and Secondary Structure of Proteins

After baseline subtraction we are ready to analyze the data. Each of the three basic secondary structures of a polypeptide chain(helix, sheet, coil) show acharacteristic CD spectrum. Aprotein consisting of theseelements should thereforedisplay a spectrum that can be deconvoluted into the individual contributions.

CD of Proteins – Near UV region

PHENYLALANINES TYROSINES TRYPTOPHANS S-S BONDS

Small extinction coefficient due to high symmetry.

Lower symmetry and hence intense absorption bands.

Has the most intense absorption band.

It has a very broad band.

Absorption maxima at 254, 256, 262 and 267 nm.

Absorption maxima at 276 nm; hydrogen bonding to the –OH group leads to a red shift of up to 4nm

Absorption maxima is 282 nm

Absorption maxima ranges from 250 – 300 nm

CD in biochemistry

Used in the understanding of the higher order structures of chiral macromolecules such as proteins and DNA. The reason for this is that the CD spectrum of a protein or DNA molecule is not a sum of the CD

spectra of the individual residues or bases, but is greatly influenced by the

3-dimension structure of the macromolecule itself. Each structure has a specific circular dichroism

signature, and this can be used to identify structural elements and to follow changes in the structure of

chiral macromolecules.

The most widely studied circular dichroism signatures are the various secondary structural elements of proteins such as the α-helix and the β sheet. This is understood to the point that CD spectra in the far-UV (below 260nm) can be used to predict the percentages of each secondary structural element in the structure of a protein.

There are many algorithms designed for fitting the circular dichroism spectra of proteins to provide estimates of secondary structure. The protein secondary structure CD analysis software distributed with the Chirascan is CDNN.

Other Applications

• CD is a particularly powerful tool to follow dynamic changes in protein structure which may result due to the effect of changing temperature, pH, ligands, or denaturants etc.

• Circular dichroism can be used to follow the kinetics of refolding of the secondary structure of a protein using changes in denaturant concentration.

• It can also be used to follow the unfolding of proteins by thermal denaturation.

• A powerful application of circular dichroism is to compare two macromolecules, or the same molecule under different conditions, and determine if they have a similar structure. This can be used simply to ascertain if - i) a newly purified protein is correctly folded, ii) determine if a mutant protein has folded correctly in comparison to the wild-type, or iii) for the analysis of biopharmaceutical products to confirm that they are still in a correctly folded active conformation.

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