Title: Circular Dichroism CD and Optical Rotatory Dispersion ORD Spectroscopies
1Circular 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)
2Plane polarized light
3Superposition of two plane polarized waves, with
orthogonal polarizations, but in phase with each
other
4Superposition of two plane polarized waves, with
orthogonal polarizations, but with a phase shift
of 90 between them
R
L
5Superposition of R L circularly polarized waves
of equal amplitudes results in linearly polarized
light
6Elliptically 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
7Absorption of linearly polarized light
8Absorption of circularly polarized light
9Effect of the refractive index of the medium on
linearly polarized light
- v c / n n refractive index
10Effect of the refractive index of the medium on
circularly polarized
11Optical 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
12Effect 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
13Optical 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
14Effect 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
15Simultaneous effect of Dn and De on linearly
polarized light
- De leads to ellipticity and Dn to a rotation of
the axes of ellipticity
16Circular 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!
17CD spectra are generally easier to interpret than
ORD spectra
CD
ORD
18Study 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
19Case 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,
20Main 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