Optical Rotation, Optical Rotary Dispersion, Circular

Birefringence and Circular Dichroism

Part I: Some Useful TheoryPart II: Applications

• Circular dichroism is used to gain information about the secondary structure of proteins and polypeptides in solution.

Biological Molecules Are Optically Active• Molecules are said to be

“optically active” (chiral) when they interact different with left and right circularly polarized light (or if they rotate the plane of polarization of light).

• Most biological molecules are optically active.

• Asymmetric chromophores (uncommon) or symmetric chromophores in asymmetric environments will interact differently with right- and left-circularly polarized light

• can result from chiral molecules such as the peptide backbone of proteins, a non-chiral molecule covalently attached to a chiral molecule (aromatic amino acid side chains), or a non-chiral molecule in an asymmetric environment (e.g., a chromophore bound to a protein).

E electric field vector that perpendicular to the direction of propagation of wave.

Unpolarized-is random polarization (all directions)

Plane polarized--magnitude varies sinusoidally in a single direction.

Circular polarized--vector magnitude is constant and traces out a circle.

• The electric field vector can oscillate randomly (unpolarized) in a plane or in a ellipse (circle).

Where is the turtle?Polarized light produced by unpolarized sunlight hitting the surface of water causes “glare” (polarized light we see as glare). Using a polarized glasses we can rid the glare (right).

Polarized light can be created using commercially-available optics called wave-plates.

Circularly Polarized Light sweeps out a helix as it propagates, or a circle (ellipse) at a point in space.

Right-Handed spins rightLeft-Handed spins left

The magnitude of the electric-field vector is constant! It does not vary sinusoidally as it does in plane polarized light.

Direction of propagation

Ehttp://www.enzim.hu/~szia/cddemo/edemo0.htm

Highly recommended to understand what birefringence, optical rotation, circular dichroism and ellipticity are, and how to visualize the phenomena.

• Birefringence refers to the direction-dependent index of refraction

• Dichroism is used to denote direction-dependent light absorption.

• Linear dichroism refers to the differential absorption of light polarized parallel or perpendicular to the some reference axis.

• Circular dichroism refers to the differential absorption of left and right circularly polarized light.

Some Confusing Terminology

Light source

Plane Polarized

Light

Analyzing Filter polarizer which is

rotated by observer until no light passes

giving angle of rotation

Sample tube containing optically active material which rotates the plane

of polarized light.

PolarizerUnpolarized

Light

Observer

Chiral molecules (asymmetric) cause the speed of EL and ER to change rotating the plane of polarization when recombined. This is called circular birefringence.

Optical rotation depends on the wavelength. When rotation is plotted vs ! we call it optical rotary dispersion.

!

Ref

ract

ive

Inde

x

absorption

In regions of an absorption band the refractive index experiences “anomalous dispersion”.

Anomalous Dispersion

Normal Dispersion

Rotation of Plan of Polarizationdue to birefringence (phase shift betweenleft and right circularly polarized light butabsorption the same.

Change in EllipticityDirectly related to differential absorption of left and right circularlypolarized light.

ORD rotates the polarization of light, CD we measure the difference in absorption of left and right circularly light.

Circular birefringence does not change the amplitude of the two circularly polarized beams (which means that the polarized plane is rotated only).

α =π

λ(nL − nR)l

[α]Tλ =α

lc

angle of rotation n(!)

specific rotation

[Θ]Tλ =[α]Tλ100

molar rotation

Rotation to the right is called dextrorototary (+). Rotation to the left is levorotary(-)

Circular dichroism is the alteration of circularly polarized light due to differences in molar absorptivity "L and "R of circularly polarized light components.

Linear polarized is the superposition of opposite circular polarized light of equal amplitude and phase

Different absorption of the left and right hand polarized component leads to change in amplitude of one arm and a change from a circle to an ellipse.

EL ER

!

• Circular Birefringence--is a wavelength dependent differential refractive index for left and right circulary polarized light in a sample. The net effect is a phase shift in either component, but NO AMPLITUDE CHANGE. The effect as a function of wavelength is observed as a rotation of plane of polarized light--called optical rotary dispersion.

• Circular Dichroism: is a wavelength dependent differential absorption for left and right circulary polarized light in a sample. The net effect is a phase shift WITH AMPLITUDE CHANGE.

ORD rotates the polarization of light, CD we measure the difference in absorption of left and right circularly light.

n refractive index " wavelength of light # angle of rotation

Light source

Light source

Sample

Sample

Preferential Absorption

LeftRight

DetectorAnalyzer

Positive rotation

Circular Polarizer Circular

Polarized Light

Detector

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.

ORD (top) and CD Spectra (bottom)

ORD

CD

ORD (top) and CD Spectra (bottom)

Circular dichroism is 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.

CD = AL - AR

AL

AR dich

rois

m

abso

rptio

n

wavelength (nm)wavelength (nm)

ORD

CD

Equations For ORD and CD Circular Dichroism• measures differences in the light absorption of left-handed

polarized light vs right-handed polarized light which arises 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

ORD and CD Reveal Asymmetric Arrangements of Chemical Groups

Absorption CD

ORD

Circular Dichroism

Part II. CD spectra of Protein

ORD and CD Can Reveal Conformations of Proteins in biological system

๏ Determination of secondary structure of proteins that cannot be crystallized

๏ 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

Applications of CD in Structural Biology Spectral Regions of Interest is the same as where UV-Vis absorption bands are in peptides and chromophores.

Amide Chromphore

• n ! !* centered around 220 nm

• ! ! !* centered around 190 nm

n -> !* involves non-bonding electrons of O of the carbonyl;

CD Reveals Asymmetric Arrangements of Chemical Groups

Different Secondary Structure Produce Unique Spectra

Dichroweb Three basic structures of proteins show a characteristic CD spectrum

Secondary structure is picked up CD with high sensitivity.

Three basic structures of proteins show a characteristic CD spectrum

Ligand-Substrate Binding

Can You Tell Which is hemoglobin? Monitoring Changes in Secondary structure

Denaturation of a Protein (unfolding) Conformational Changes Using CD

Conformational Changes Using CD Conformational Changes Using CD

Monitoring Kinetics Secondary Structure Determination

Fitting CD Spectra To Estimate Conformation Fitting CD Spectra To Estimate Conformation

Fitting CD Spectra To Estimate Conformation

Circular DichroismPart III: Nucleic Acids Applications

Structure of DNA

A-DNA B-DNA Z-DNA

• Most common DNA conformation in vivo

• Narrower, more elongated helix than A.

• Wide major groove easily accessible to proteins

• Narrow minor groove

• Most RNA and RNA-DNA duplex in this form

• Shorter, wider helix than B.• Deep, narrow major groove not

easily accessible to proteins• Wide, shallow minor groove

accessible to proteins, but lower information content than major groove.

• Favored conformation at low water concentrations

A-DNA

B-DNA

Z-DNA

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.

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 first observation of Z-DNA was made with CD in 1972.

Chromophores of Nucleic Acid

Absorption Spectra CD Spectra

Chromophores In Nucleic Acid

• ! ! !* transitions begin about 300 nm

• n ! !* buried under ! ! !* 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.

Native DNA

Denatured Native DNA

Average spectrum for the four component deoxynucleotides

•CD occurs only where normal absorption occurs

•CD is more complicated revealing bands that not separated in the normal absorption spectrum

CD vs. Absorption (Melting Curve)

The CD of poly(dA) poly(dT) as a function of temperature

Temperature Effect on DNA

10 C

38.80 C

44.70 C

58.30 C48.20 C

CD is sensitive to the change in conformation when DNA melts with increasing temperature

CD of double-stranded DNA and RNA

Polymer

dimer

monomer

CD of single stranded oilgo(rA) in aqueous solution at pH 7

Formation of helical structure is a super asymmetry that gives rise to degenerate interactions between chromophoric bases and results in intense CD spectra

From monomer to polymer

Solvent Effect on DNA Structure I

Calf thymus DNA

25% methanol50% methanol65% methanol75% methanol

0% methanol

95% methanol

10.2 base pair B-form

10.4 base pair B-form

Solvent Effect on DNA Structure II

65% methanol

70% methanol

75% methanol

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