CIRCULAR
Circular dichroism (CD) spectroscopy is a form of
light absorption spectroscopy that measures the
difference in absorbance...
Important Basic concepts

• Definitions of asymmetry
• What is optical Activity
• What is polarized light
Circular Dichroism (CD) phenomena that result when
asymmetrical molecules interact with plane polarized light.

Asymmetry
...
Light

Polarized Light
Circular Polarized Light
Instrumentation

Prism Polarizer
Nitrogen purging

• The function of purging the CD instrument with
nitrogen is to remove oxygen from the lamp
housing, mon...
Sample preparation and measurement
• Additives, buffers and stabilizing compounds: Any

compound which absorbs in the regi...
•
•
•
•

Protein Concentration: 0.5 mg/ml
Cell Path Length: 0.5 mm
Stabilizers (Metal ions, etc.): minimum
Buffer Concentr...
Cutoff Wavelengths For Common Solvents and
Buffers
IL-IR

IL-IR
• The molar ellipticity [ ] is related to the difference
in extinction coefficients
[ ] = 3298 Δε.
• Here [ has the standa...
Result Table
Resultant Spectra

CD spectra of calf thymus DNA titrated with BPSQ; at (a) r 0 ¼ 0; (b) 0.08;
(c) 0.16; (d) 0.24; (e) 0.4...
Applications of CD spectroscopy
A. Estimation of protein and nucleic acid
conformation

B. Determination of conformational...
D. Determination of binding constants by:
1. direct titrations
2. serial dilutions of complexes
3. changes in stability to...
• It has been shown that CD spectra between 260
and approximately 180 nm can be analyzed for
the different secondary struc...
• For proteins we will be mainly concerned with
absorption in the ultraviolet region of the spectrum
from the peptide bond...
Resultant Spectra
Chromophore Analysis
CD Spectra of Protein secondary
Structures
-1

] x10-3 degrees cm2 dmol
• In a first approximation, a CD spectrum of a protein
or polypeptide can be treated as a sum of three
components: -helica...
• As we have three unknowns in this equation, a
measurement at 3 points (different wavelengths)
would suffice to solve the...
Resultant Spectra
Resultant Spectra
http://www.cryst.bbk.ac.uk/cdweb/html/home.html
• CD bands in the near UV region (260 – 350 nm)
are observed in a folded protein where aromatic
side chains are immobilize...
Peptide binding to nucleic acids
Protein-protein interactions

CD spectra before and after mixing SCM and Mj HSP16.5.
• Circular dichroism spectroscopy is used to gain
information about the secondary structure and
folded state of proteins a...
Drawbacks:
• Interference with solvent absorption in the UV
region
• Only very dilute, non-absorbing buffers allow
measure...
Circular Dichroism ppt,
Circular Dichroism ppt,
Circular Dichroism ppt,
Circular Dichroism ppt,
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Transcript of "Circular Dichroism ppt, "

  1. 1. CIRCULAR
  2. 2. Circular dichroism (CD) spectroscopy is a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance. Jasco J-810 Circular Dichroism System
  3. 3. Important Basic concepts • Definitions of asymmetry • What is optical Activity • What is polarized light
  4. 4. Circular Dichroism (CD) phenomena that result when asymmetrical molecules interact with plane polarized light. Asymmetry Optical activity
  5. 5. Light Polarized Light
  6. 6. Circular Polarized Light
  7. 7. Instrumentation Prism Polarizer
  8. 8. Nitrogen purging • The function of purging the CD instrument with nitrogen is to remove oxygen from the lamp housing, monochromator, and the sample chamber. • The presence of oxygen is detrimental for two reasons. When deep ultraviolet light strikes oxygen, ozone is produced. Ozone causes degradation of optics and can cause respiratory problems. The second reason for removing oxygen is that oxygen absorbs deep UV light, thus reducing the light available for the measurement.
  9. 9. Sample preparation and measurement • Additives, buffers and stabilizing compounds: Any compound which absorbs in the region of interest (250 –190 nm) should be avoided (see below). • Protein solution: The protein solution should contain only those chemicals necessary to maintain protein stability, and at the lowest concentrations possible. Any additional protein or peptide will contribute to the CD signal. • Data collection: Initial experiments are useful to establish the best conditions for the "real" experiment. Cells of 0.5 mm path length offer a good
  10. 10. • • • • Protein Concentration: 0.5 mg/ml Cell Path Length: 0.5 mm Stabilizers (Metal ions, etc.): minimum Buffer Concentration : 5 mM or as low as possible while maintaining protein stability
  11. 11. Cutoff Wavelengths For Common Solvents and Buffers
  12. 12. IL-IR IL-IR
  13. 13. • The molar ellipticity [ ] is related to the difference in extinction coefficients [ ] = 3298 Δε. • Here [ has the standard units of degrees cm2 dmol -1 • The molar ellipticity has the units degrees deciliters mol-1 decimeter-1.
  14. 14. Result Table
  15. 15. Resultant Spectra CD spectra of calf thymus DNA titrated with BPSQ; at (a) r 0 ¼ 0; (b) 0.08; (c) 0.16; (d) 0.24; (e) 0.40; and (g) 0.72.
  16. 16. Applications of CD spectroscopy A. Estimation of protein and nucleic acid conformation B. Determination of conformational changes due to the interactions of asymmetric molecules e.g. 1. Protein-protein interactions 2. Protein-DNA interactions 3. Protein-Ligand interactions 4. DNA-Ligand interactions C. Determination of the thermodynamics of folding and unfolding of proteins and nucleic
  17. 17. D. Determination of binding constants by: 1. direct titrations 2. serial dilutions of complexes 3. changes in stability to thermal or chemical denaturation E. Kinetics of macromolecules folding and unfolding of
  18. 18. • It has been shown that CD spectra between 260 and approximately 180 nm can be analyzed for the different secondary structural types: alpha helix, parallel and anti-parallel beta sheets, turns, and other. • A number of excellent review articles are available describing the technique and its application (Woody, 1985 and Johnson, 1990). • Modern secondary structure determination by CD are reported to achieve accuracies of 0.97 for helices, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types (Manavalan & Johnson, 1987).
  19. 19. • For proteins we will be mainly concerned with absorption in the ultraviolet region of the spectrum from the peptide bonds (symmetric chromophores) and amino acid sidechains in proteins. • Protein chromophores can be divided into three classes: the peptide bond, the amino acid sidechains, and any prosthetic groups. • The lowest energy transition in the peptide chromophore is an n → * transition observed at 210 - 220 nm with very weak intensity ( max~100). -------n ---- → n→ 90 nm max~7000 nm max~100
  20. 20. Resultant Spectra
  21. 21. Chromophore Analysis
  22. 22. CD Spectra of Protein secondary Structures
  23. 23. -1 ] x10-3 degrees cm2 dmol
  24. 24. • In a first approximation, a CD spectrum of a protein or polypeptide can be treated as a sum of three components: -helical, -sheet, and random coil contributions to the spectrum. • At each wavelength, the ellipticity (θ) of the spectrum will contain a linear combination of these components: (1) • θT is the total measured ellipticity, θh the contribution from helix, θs for sheet, θc for coil, and the corresponding χ the fraction of this contribution.
  25. 25. • As we have three unknowns in this equation, a measurement at 3 points (different wavelengths) would suffice to solve the problem for χ, the fraction of each contribution to the total measured signal. • We usually have many more data points available from our measurement (e.g., a whole CD spectrum, sampled at 1 nm intervals from 190 to 250 nm). In this case, we can try to minimize the total deviation between all data points and calculated model values. This is done by a minimization of the sum of residuals squared (s.r.s.), which looks as follows :
  26. 26. Resultant Spectra
  27. 27. Resultant Spectra
  28. 28. http://www.cryst.bbk.ac.uk/cdweb/html/home.html
  29. 29. • CD bands in the near UV region (260 – 350 nm) are observed in a folded protein where aromatic side chains are immobilized in an asymmetric environment. • The CD of aromatic residues is very small in the absence of ordered structure (e.g. short peptides). • The signs, magnitudes, and wavelengths of aromatic CD bands cannot be calculated; they depend on the immediate structural and electronic environment of the immobilized chromophores. • The near-UV CD spectrum has very high sensitivity for the native state of a protein. It can
  30. 30. Peptide binding to nucleic acids
  31. 31. Protein-protein interactions CD spectra before and after mixing SCM and Mj HSP16.5.
  32. 32. • Circular dichroism spectroscopy is used to gain information about the secondary structure and folded state of proteins and polypeptides in solution. Benefits: • Uses very little sample (200 ul of 0.5 mg/ml solution in standard cells) • Non-destructive • Relative changes due to influence of environment on sample (pH, denaturants, temperature, etc.) can be monitored accurately.
  33. 33. Drawbacks: • Interference with solvent absorption in the UV region • Only very dilute, non-absorbing buffers allow measurements below 200 nm • Absolute measurements subject to a number of experimental errors • Average accuracy of fits about +/- 10% • CD spectropolarimeter is relatively expensive
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