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Bios 532
Circular Dichroism
Circular dichroism is a form of
chiroptical spectroscopy.
Chiroptical spectroscopy uses circularly
polarized light, and commonly exploits
differences in the interactions of
asymmetric chromophores with left- and
right-circularly polarized light.
Propagates as a plane wave = linear polarization
Two plane waves, equal amplitude, phase is 90 = circular polarization
Two plane waves of differing amplitude, phase is 90 OR
Two plane waves, equal amplitude, phase ≠ 90 = elliptical polarization
natural light = unpolarized, propagates in all planes
The relationship between CD signal and the
Beer-Lambert Law
Alcp = lcplc
for left circularly polarized light (lcp)
The definition of CD is
A = Alcp - Arcp = lcplc - rcplc =  lc
Where the difference in absorbance measured is equivalent to
, the decadic molar CD.
The absorbance change in CD experiments
is very small. Modern instruments can
measure this value directly, but historically,
CD was measured in terms of ellipticity.
In the biochemical sciences, CD is commonly
still expressed in terms of molar ellipticity, .
The relationship between molar ellipticity and
the change in absorption coefficient, , is:
[] = 3298 ∆
A superposition of vectors of rightand left- circularly polarized light of
equal amplitude and phase
represents linearly polarized right.
When an optically active sample
differs in its absorbance for the right
vs. left circular light, the resultant
amplitude of the more strongly
absorbed component will be smaller
than that of the less absorbed
component. The consequence is that
a projection of the resulting
amplitude now yields an ellipse
instead of the usual line.
http://www-structure.llnl.gov/cd/cdtutorial.htm
CD is used for proteins because of the chiral
nature of the structural features of proteins Biopolymers are intrinsically asymmetric;
L-amino acids predominate over D-amino acids.
L-alanine
D-alanine
Images from onlinediscussion of amino acid chirality:
http://opbs.okstate.edu/~Blair/Bioch2344/Chapter7/Chapter7.htm
Amino acids
Chromophores PHE, TRP, TYR When an aromatic residue is held rigidly in space,
its environment is asymmetric, and it will exhibit circular dichroism.
Amide bond
In secondary structure conformations, the backbone and
the amide bond chromophores are
arranged in regular, organized, asymmetric patterns.
Near UV CD (250 - 350 nm)
Near-UV CD spectroscopy is dominated
by Phe, Tyr, Trp and disulfides
Far UV CD (180 - 250 nm)
The amide group is the most abundant
CD chromophore in proteins.
* transition
n* transition
~ 190 nm
~220 nm
Far UV CD
exhibits distinct
spectra for
-helical,
-sheet,
and random coil
secondary structure.
Brief CD tutorial online: http://www.cryst.bbk.ac.uk/cdweb/html/info_cd.html
A more detailed tutorial: http://www.newark.rutgers.edu/chemistry/grad/chem585/lecture1.html
CD exhibits characteristic spectra for protein secondary structure
features - alpha helix, beta sheet random coil
CD spectra can be deconvoluted using a set of basis spectra, but the
analysis is difficult and contains bias dependent on the choice of
reference spectra.
Primary uses for CD:
• analyze structural changes in a protein upon some perturbation
• compare the structure of a mutant protein to the parent protein
• screen candidate proteins for more detailed structural analysis
(NMR or X-ray crystallography)
apo-myoglobin
Green Fluorescent Protein
Structure of Lac Repressor