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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 right-
and 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.

  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:
                          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   ~ 190 nm
         n* transition   ~220 nm
                                              Far UV CD
                                            exhibits distinct
                                              spectra for
                                            and random coil
                                          secondary structure.
        Brief CD tutorial online:
A more detailed tutorial:
 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)
Green Fluorescent Protein
Structure of Lac Repressor

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