Circular Dichroism CD and Optical Rotatory Dispersion ORD Spectroscopies

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Circular Dichroism (CD) and Optical Rotatory
Dispersion (ORD) Spectroscopies

• Physical Biochemistry November 2006
• Dr Ardan Patwardhan a.patwardhan_at_imperial.ac.uk
  Faculty of Natural Sciences Imperial College
  London
• Animations taken from Dr Andras Szilagyi
  (http//www.enzim.hu/szia/cddemo/edemo0.htm)

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Plane polarized light
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Superposition of two plane polarized waves with
orthogonal polarizations but in phase with each
other
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Superposition of two plane polarized waves with
orthogonal polarizations but with a phase shift
of 90 between them
R
L
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Superposition of R L circularly polarized waves
of equal amplitudes results in linearly polarized
light
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Elliptically polarized light

• Is what you get when you combine
• 2 linearly polarized waves () of equal
  amplitudes that are phase shifted by any angle
  other than 0 or 90
• 2 linearly polarized waves () of unequal
  amplitudes that are phase shifted by any angle
  other than 0
• R L circularly polarized waves of unequal
  amplitudes

Unequal amplitudes 90 phase shifted
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Absorption of linearly polarized light
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Absorption of circularly polarized light
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Effect of the refractive index of the medium on
linearly polarized light

• v c / n n refractive index

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Effect of the refractive index of the medium on
circularly polarized
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Optical activity

• Enantiomers are optically active
• An asymmetric environment can also confer optical
  activity to a molecule
• Optically active molecules have different
  refractive indices and different extinction
  coefficients for L and R circularly polarised
  light

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Effect of different refractive indices circular
bifringence
But as they travel through the medium at
different speeds they will be phase shifted when
they exit the medium
The superposition of the two circularly polarized
components is still a linearly polarized wave
the phase shift manifests itself as a rotation of
the polarization plane
Consider a linearly polarised wave as a
superposition of R L circularly polarized waves
Initially these waves are in phase
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Optical Rotatory Dispersion (ORD) Spectroscopy

• The refractive indices for R and L varies as a
  function of wavelength as does the difference
  between them
• One can measure Dn by measuring the rotation of
  linearly polarized light

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Effect of different e circular dichroism
Consider a linearly polarised wave as a
superposition of R L circularly polarized waves
The two circularly polarized waves are attenuated
by different amounts by the specimen
The wave exiting the specimen is the
superposition of L and R waves with different
amplitudes i.e. elliptically polarised
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Simultaneous effect of Dn and De on linearly
polarized light

• De leads to ellipticity and Dn to a rotation of
  the axes of ellipticity

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Circular Dichroism (CD) Spectroscopy
To examine the circular dichroism of an optically
active specimen illuminate it directly with
circularly polarized light of alternating
polarizations
The spectrum obtained is almost identical to an
absorption spectrum except that the peaks can be
both positive and negative!
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CD spectra are generally easier to interpret than
ORD spectra
CD
ORD
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Study of proteins with CD

• Peptide bond absorption (200nm) mainly studied
  (asymmetry imposed by environment)
• CD good for determining alpha helical content
• Excellent at monitoring conformational changes-
  changes in enzyme structure upon binding-
  protein denaturation- protein folding

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Case Study

• CD spectra of Bcl-2glutathione S-transferase
  fusion protein with (solid line) and without
  (dotdashed line) Taxol added.
• Before this study it was not known that the drug
  bound to this protein. A large conformational
  change involving a loop region occurs and can
  be monitored at the peak wavelength (solid arrow)
  for screening with changes in the isosbestic
  point (dashed arrow) monitored to eliminate false
  positives. Adapted from the Journal of Molecular
  Biology vol. 285 D.J. Rodi R.W. Janes H.J.
  Sanganee R.A. Holton B.A. Wallace and L.
  Makowski Screening of a library of
  phage-displayed peptides identifies human bcl-2
  as a Taxol-binding protein pp. 197203

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Main Points

• Optically active absorbing chromophores present
  different extinction coefficients for R and L
  circularly polarized waves
• CD spectroscopy exploits this phenomena to probe
  the handedness of the environment of the
  chromophores
• The technique is good at estimating alpha helical
  content and at studying dynamic changes is
  secondary structure