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CD Spectroscopy Presentation1.pptx
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- 2. Why use CD? 1.Information on molecular and electronic structure. 2. Experiments are relatively quick and easy to perform. 3. Non-destructive (can recover most samples). Advantages Simple and quick experiments No extensive preparation Measurement on solution phase Any size of macromolecule Microsecond time resolution Relatively low concentration /amounts of sample Figure: Advantages of CD Spectropolarimeter 2 4. Solution phase. Crystallization process could change molecular structure. 5. Low concentrations (0.1 mg/mL). Doesn’t require a concentration which could change the system being studied.
- 3. Circular dichroism isthe difference in the absorption of left-handed circularly polarized light (L-CPL) and right-handed circularly polarized light (R-CPL). Occurs when a molecule contains one or more chiral chromophores. • Intrinsically chiral • Covalently linked to chiral center • Placed in asymmetric environment Circular dichroism = ΔA(λ) = A(λ) LCPL - A(λ) RCPL (where λ is the wavelength) What is Circular Dichroism? 3 Cordierite
- 4. Chiral molecules https://socratic.org/organic-chemistry-1/r-and-s-configurations/chiral-and-achiral-molecules-1 N CC H H R O O H Side Chain Carboxyl Group H Amino Group Figure: General Structure of Amino Acid Chira l Zwitter Ion Polarity dependent on side chain Change in protein structure alters the polarity 4
- 5. Electromagnetic wave http://www.emagtech.com/wiki/index.php?title=File:EM_wave.png Light path Amplitude Amplitude:intensity of wave from tip of crest to central axis Wavelength: distance between two consecutive crests Polarization: directionality of electric and magnetic fields 5 x z y Unpolarise d A Polarization of Light y x z Linearly or Plane Polarised B Left Handed Right Handed Circularly Polarised C Figure: Schemes of the electric field components of unpolarized.
- 6. Left CircularlyPolarized lightRightCircularlyPolarized light 6 Verticallypolarizedlight Horizontallypolarized light
- 7. ∆𝐴 = 𝐴𝐿− 𝐴𝑅 LH RH Elliptically polarized light Elliptically polarized light: electric field components rotate along beam propagation, perpendicular, unequal in amplitude, 90° phase difference Circularly polarized light: electric field components rotate along beam propagation, perpendicular, equal in amplitude, 90° phase difference 7
- 8. CircularDichroism • Some materialspossessaspecialProperty.Assume that a plane-polarizedlight wave (blue)traversesa mediumthat does not absorb the left circularly polarized component (red) of the wave at all but highly absorbs the right circularly polarizedcomponent(green). Theintensity of the greencomponentdecreasesIn comparisonto the redone. The superposition of the two components yields a resulting field vector that rotates along an ellipsoid path and is called an elliptically polarizedlight. 8 Optically active molecules absorb left handed and right handed light differentially. This difference in absorbance is called CIRCULAR DICHROISM or CD. Optically active molecule s Sampl e Left Handed Right Handed Circularl y Polarise d Figure: Schematic Representation of Circular Dichroism
- 9. JASCO Corporation Product JASCOCorporation Hachioji, Tokyo Since 1958 9
- 10. Principleof Measurement Light Source Unpolarize dlight Linearly polarized light PEM Circularly polarized light Double Monochromator Sample PMT Detector RCP LCP RCP LCP Prisms 10
- 11. CD Spectrum 100 nm 950 nm Near IR 750-950 nm UV-Vis 350-750 nm NearUV 250-350 nm Vacuum UV 100- 200 nm Far UV 200-250 nm Amide , Secondary Structure Aromatic groups, Tertiary structure, Folding Extrinsic groups, Tertiary structure, Ligand binding Figure: Schematic Representation of Classification of CD Spectrum 11
- 12. CD Spectra ofProtein Secondary Structures -ve band (nm) +ve band (nm) α-helix 222 192 208 β-sheet 216 195 Random coil 200 Figure: Fasman Standard Curve for Polylysine Reference: Ranjbar B, Gill P. Circular Dichroism Techniques: Biomolecular and Nanostructural Analyses- A Review. Chemical Biology & Drug Design. 2009;74(2):101-120. Table 1: Features of CD Spectra of Protein Secondary Structures 12
- 13. Far-UV CD spectraof protein solutions -sheet turn Random coil -helix 13 Near-UV CDspectraofproteinsolutions Kelly, et al. Biochimica et Biophysica Acta 1751 (2005). http://www.slideshare.net/kleppingerb/lipids-and-proteins
- 14. CD Multivariate SSEProgram A calibration model for secondary structure abundance ratios obtained by X-ray crystallography and CD is produced using PCR or PLS, which is then used to estimate the unknown secondary structure of proteins. 14
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Editor's Notes
- #2 This is very interesting topic for Chemistry as well as biological students. So, What is circular Dichroism is a type of spectroscopy process to understand the structure of macromolecules and also charged particles and how the charging and discharging occurs? Actually, It is vigorously used in understanding the structure of proteins, understanding the Secondary structure of any chiral molecule or optical active molecule.
- #3 Simple and quick experiment give resolution in microseconds.
- #4 Phenomena of Circular dichroism derived from French word Cordierite which was one of the first mineral which exhibited Circular Dichroism. So, For understanding circular dichroism we understand to know that how light moves into different ways. The movement of light in the form of waves due to two vectors.
- #5 Superimposed Chiral:- Molecules in a given material that absorb particular wavelength of visible light or light energy and in doing so confer a color on the material. Polarity dependent on side chain. Change in protein structure alters the polarity. Example of C
- #6 Linearly Polarized light occurs when electric fields vectors oscillates only in one plane. That’s why it is called the polarized light because all the other oscillations are getting cancelled to some reasons. Light is produced due to two vectors. Electric and magnetic fields are perpendicular to each other have magnitudes. They are labelled as red and blue colour are vibrating perpendicular to each other and in one direction. So the important thing for linearly polarized light is that its magnitude varies but direction of vibration remains constant. Now in contrast to linearly polarized we have circularly polarized light which is actually used for circular dichroism. In circularly polarized light we have constant magnitude and but the direction is varies. Actually the direction is such that there are two fields one upon ¼ lamda out of phase with each other. So, They are rapidly rotating, make oscillating magnetic fields which is rotating.
- #7 Here is the nice animation of linearly polarized light that further elaborating my point is vibrating back and forth. So, magnitude varying but the direction is constant. So, this is linearly polarized light. While Here is the circularly polarized light you can see it is making a twist around imagenery axis because the both the fileds which are labelled here as red and green. They are out of sink with each other about quarter of the wavelength. And that’s why it makes this twist. Now this is the foundational principle of Circular Dichroism.
- #8 Here is the optical rotation which is shown by a chiral molecule that canot be superimposed on each other like hands. When circularly polarized light exposed to optical active substance, it will rotate the light by specific degree and this is called specific rotation. Alpha Rotation It really depends upon the chiral molecule. And its leads to elliptically polarized light bcz of the difference btw the rotation.
- #9 Degree of ellipticity is actually the measure of circular dichroism. Schematic representation to understand is
- #10 J-1500 called CD Poarimetry Firstly made by JASCO Compnay in 1958 located in Tokyo.
- #11 If we look at the basic understanding of the circulalrly dichroism we can see here Here it is the circularly polarized light. It is consisting of both the directions. Both the lights are travelling because it is a photon beam that is travelling from one place towards a particular direction. Now this is the optically active sample (we don’t know the structure of that molecule and want to know the structure of the molecule. So, put the sample in there. As the photon beam passing through that sample the sample is changing this polarization pattern that’s what detected by the detector machine. For example here we can see that photon beam left handed and right handed polarized light completely equally participating in here. But now once it is passing through the optically active molecule that in that case seen the left handed polarized light is very very short. This means that the protentional absorption of left handed light because LCP light is absorbed by the sample. By looking at this patteren detector draw a graph connected with the computer. This is acutually the schematic presentation of CD.
- #12 Peptides bond absorb less than 240nm. Aromatic Amino acids absorb 260-320nm. Like tryptone. Tyrosine. Phenylalanine. Disulfide bond absorb around 260nm. There are different spectra for different cofactors. Flavin molecules show absorbance of 300-500nm. Heme 410nm.
- #13 Degree of ellipticity is actually the measure of circular dichroism. Fasman standard spectra Here is the example of CD to how to characterize the structure of protein. Look at the ellipticity denoted by theta is here. We can see that protiens having different kinds of absorption spectrum. For example this black line show alpha helix.
- #14 So, we compare our protein of interest with this standard spectra.
- #15 Circular Dichroism Applications Structural Characterization of proteins Antibody stability evaluation Thermal stability study DNA and G-quadruplex structure Stopped flow folding study Microassay methods
- #16 What is the word birefringence? the refraction of light in an anisotropic material in two slightly different directions to form two rays. also : the visual effect produced by birefringence. What is an example of birefringence? This is a very interesting (and often undesirable) behavior of some materials that we called birefringence. For example, let's say that you have a piece of quartz and you look at a text behind it. Under certain conditions, it will be possible to see the same text twice, kind of like a ghost image of the original text. MonoChomator? We need a polarizer to make light polarize in particular direction. So we used only one type of source that is a monochromatic light consisting of only one particular wavelength. So, we look at here monochromatic light passes through PEM and through opticle active molecule detected by PMT detector