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The electromagnetic radiation The electromagnetic radiation is a complex wave form that can be considered to behave as two simple wave motions at right.

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Presentation on theme: "The electromagnetic radiation The electromagnetic radiation is a complex wave form that can be considered to behave as two simple wave motions at right."— Presentation transcript:

1 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:

2 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.

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4 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. E R = Right-handed circularly polarized light E L = Left-handed circularly polarized light

5 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. E R = Right-handed circularly polarized light E L = Left-handed circularly polarized light k is the absorbtion coefficient defined as I =I 0 x10 -kd I and I 0 are the intensity of incident and resultant light, d is the cell thickness

6 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 E R = Right-handed circularly polarized light E L = Left-handed circularly polarized light n is the refractive index Circularly-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).

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8 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)

9 At a given wavelength: ΔA = (A L - A R ) 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 = (ε L - ε R ) C l Where ε L and ε R are the molar extinction coefficients for RCP and LCP light C is the molar concentration l is path length Then Δε = (ε L - ε R ) - is the molar circular dichroism Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. The ellipticity is: θ(gradi) = 32.98° Δε CD Units

10   tan  = b/a

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12 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 =

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20 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.Advantages

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22 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 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.

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24 Far-UV CD for determining protein structure 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)

25 Far-UV CD for determining protein structure 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->  *)

26 Examples Azurin  proteinMyoglobin 80%  -helix

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30 Use far-UV CD to determine amounts of secondary 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 = f  S  ( ) + f  S  ( ) + f RC S RC ( ), where S  ( ), S  ( ), and S RC ( ) 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.

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32 Near-UV CD spectroscopy ( nm) is dominated 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 ( 1 L a and 1 L b ) that are directed in the plane of the  -bonding system and are orthogonal to each other. Phe Tyr Trp S-S Range nm Tyr 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 nm Trp has the most intense absorption band centered at 282 nm. Hydrogen- bonding to the NH can shift the 1 L a band by as much as 12 nm. Disulfide (S-S) spectra have a broad band at nm. Range nm Absorption maxima at 254, 256, 262 and 267 nm

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35 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.Summary

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44 CD spectrum of Copper-Zinc SOD d-d LMCT MO approach can be used to interpret the CD spectrum


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