The electromagnetic radiation The electromagnetic radiation The electromagnetic radiation is a complex wave form that can be considered to behave as two simple wave motions at right angles to each other. One of these waves is magnetic (M) and the other is electric (E). Electromagnetic waves are generated by oscillating electric or magnetic dipoles and are propagated at the speed of light (c). Since the E- and the M-components are always perpendicular to each other, it is sufficient, in many cases, to consider only the E-component in describing the wave:

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

Circularly polarized light Circularly polarized light The amplitude of the E-wave could oscillate in any direction perpendicular to the direction of propagation (z). Unpolarized light, the type we get from the sun or a light bulb contains oscillations of the E-components in all directions perpendicular to the direction of propagation. Linearly polarized light results when the direction of the E-component is restricted to a plane perpendicular to the direction of propagation while its amplitude oscillates. Circularly polarized light is another form of polarization - in this case, the amplitude is constant and it is the direction that oscillates.

Physics of Cirular Dichroism (CD)Physics of Cirular Dichroism (CD)Linear polarized light can be seen as the result of two equal amplitudes of opposite circular polarization. After passing through an optically active sample, circularly polarized light can be changed in two aspects. The two components are still circularly-polarized, but the magnitudes of the two counter-rotating E-components could no longer be equal as the molar extinction coefficients, and therefore the resulting absorbance, for right- and left-polarized light are different. The direction of the E-vector no longer traces a circle - instead it traces an ellipse (actually an elliptical screw if we do not confine ourselves to the projection) There will also be a rotation () of the major axis of the ellipse due to differences in refractive indices.

ER = Right-handed circularly polarized light

EL = Left-handed circularly polarized light

Physics of Cirular Dichroism (CD)Physics of Cirular Dichroism (CD)Right- and left-circularly polarized light can be absorbed at different extent at some wavelengths due to differences in extinction coefficients for the two polarized rays: this is called circular dichroism (CD).The CD curve is the dependence of L-R versus .

ER = Right-handed circularly polarized light

EL = Left-handed circularly polarized light

c

kkεε RL

RL

k is the absorbtion coefficient defined asI =I0x10-kd

I and I0 are the intensity of incident and resultant light,d is the cell thickness

Physics of Optical Rotary Dispersion (ORD)Physics of Optical Rotary Dispersion (ORD)

If linear polarized light is passed through a sample for which the refrative indices of the left and right-handed polarized components differ, the components will, upon recombination, give plane polarized radiation in which the plane of the polarization is rotated by an angle , given by

ER = Right-handed circularly polarized light

EL = Left-handed circularly polarized light

λ

nn α RL

n is the refractive indexCircularly-polarized light rays will travel through an optically active medium with different velocities due to the different indices of refraction for right- and left-circularly polarized light called optical rotation or circular birefringence. The variation of optical rotation as a function of wavelength is called optical rotary dispersion (ORD).

• Circular dichroism (CD) spectroscopy is an absorption spectroscopy that can provide information on the structure of many types of biological macromolecules.• Circular dichroism spectroscopy measures the difference in absorbance of right- and left-circularly polarized light by a substance

Cirular Dichroism (CD)Cirular Dichroism (CD)

At a given wavelength:ΔA = (AL - AR)

ΔA is the difference between absorbance of left circularly polarized and right circularly polarized light

(this is what is usually measured). it can also be expressed as: ΔA = (εL - εR) C l

Where εL and εR are the molar extinction coefficients for RCP and LCP

lightC is the molar concentrationl is path lengthThen Δε = (εL - εR)   -   is the molar circular dichroismAlthough ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity.

The ellipticity is:

θ(gradi) = 32.98° Δε

CD UnitsCD Units

tan = b/a

Mean molar residue ellipticity

Methods for estimating secondary structure in polymers, proteins and polypeptides in particular, often require that the measured molar ellipticity spectrum be converted to a normalized value, specifically a value independent of the polymer length.

Mean molar residue ellipticity is used for this purpose; it is simply the measured molar ellipticity of the molecule divided by the number of monomer units (residues) in the molecule.

d = degrees[]mr=

• CD spectroscopy requires only very small amounts of material (100 micrograms or less) and measurements can be done very quickly (in 30 minutes or less).

• CD spectroscopy in the UV region can be used to monitor: 1) secondary structure, 2) conformational changes, 3) environmental effects, 4) protein folding and denaturation, and 5) dynamics.

• CD spectroscopy in the Visible region provides an increased resolution for the electronic transition absorptions

• The information produced by CD spectroscopy is complementary to the structural information obtained by other biophysical methods such as X-ray crystallography and NMR spectroscopy, especially as it can be done under conditions which mimic those found in vivo.

AdvantagesAdvantages

Biological macromolecules such as proteins and DNA are composed of optically active elements and because they can adopt different types of three-dimensional structures, each type of molecule produces a distinct CD spectra.

CD of Proteins and DNACD of Proteins and DNA

Different types of protein secondary structures (helices, sheets turns and coils) give rise to different CD spectra:

• The wavelengths of light that are most useful for examining the structures of proteins and DNA are in the ultraviolet (UV) or vacuum ultraviolet (VUV) ranges (from 190 to 300 nm) because these are the regions of the electronic transitions of the peptide backbone and side chains in proteins and the purine and pyrimidine bases in DNA.

Far-UV CD for determining protein Far-UV CD for determining protein structurestructure

n -> * centered around 220 nm max 100

->* centered around 190 nm max

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

The intensity and energy of these transitions depends on and (i.e., secondary structure)

Far-UV CD for determining protein Far-UV CD for determining protein structurestructure

For example, the absorption spectrum of poly-L-lysine in an -helix, -sheet, and unordered (random coil) differ due to long-range order in the amide chromophore.

•Far UV-CD of random coil (RC)

positive at 212 nm (->*)

negative at 195 nm (n->*)

•Far UV-CD of -sheet

negative at 218 nm (->*)

positive at 196 nm (n->*)

•Far UV-CD of -helix

the ->* transitions leads to positive at 192 nm and negative at 209 nm

negative at 222 nm is red shifted (n->*)

ExamplesExamples

Azurin protein Myoglobin 80% -helix

Use far-UV CD to determine amounts of Use far-UV CD to determine amounts of secondary structure in proteinssecondary structure in proteins

Generate basis sets by determining spectra of pure -helix, -sheet, etc. of synthetic peptides to deconvolute CD spectra of proteins with know structures to generate basis sets of each of secondary structure

For example, poly-L-lysine {(Lys)n} can adopt 3 different conformations merely by varying the pH and

temperature:

random coil at pH 7.0-helix at pH 10.8-form at pH 11.1 after heating to 52°C and recooling

CD spectrum of unknown protein = fS() + fS() + fRCSRC(),

where S(), S(), and SRC() are derived from poly-L-lysine basis

spectra.The disadvantage of this method is that although these basis sets are easily determined by direct measurement, they do not always agree from one lab to another. In addition, chain length and aggregation affect the basis set spectra. However, this method is usually accurate to within 10% for -helix content.Usually, the deconvolution is reliable mostly for helix and to a less extent for sheet. The distinction between antiparallel and parallel beta sheets looks very difficult and somewhat hazardous.

Near-UV CD spectroscopy (250 - 350 nm) is Near-UV CD spectroscopy (250 - 350 nm) is dominated by Phe, Tyr, Trp and disulfidesdominated by Phe, Tyr, Trp and disulfides

An aromatic residue rigid in space has an asymmetric environment and it shows circular dichroism in the near-UV.In a “molten globule” state the far-UV CD is retained while the near-UV CD is lost we can discriminate between completely folded and molten globule states

Aromatics have allowed ->* transitions (1La and 1Lb) that are directed in the

plane of the -bonding system and are orthogonal to each other.

Phe Tyr Trp S-SRange 270-290 nmTyr has absorption maximum at 276 nm and a shoulder at 283 nm. Hydrogen-bonding to the hydroxyl group leads to a red-shift of up to 4 nm

Range 280-300 nmTrp has the most intense absorption band centered at 282 nm. Hydrogen-bonding to the NH can shift the 1La band by as

much as 12 nm.

Disulfide (S-S) spectra have a broad band at 250 - 300 nm.

Range 250-270 nm

Absorption maxima at 254, 256, 262 and 267 nm

•CD has an important role in the structural determinants of proteins

•The real power of CD is mainly in the analysis of structural changes in a protein upon some perturbation, or in comparison of the structure of an engineered protein to the parent protein.

•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 NMR or X-ray crystallography.

SummarySummary

CD spectrum of Copper-Zinc SOD CD spectrum of Copper-Zinc SOD

d-d

d-d

d-d

LMCTLMCT

MO approach can be used to interpret the CD spectrum