bios 532 circular dichroism. circular dichroism is a form of chiroptical spectroscopy. chiroptical...

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 polarizationTwo 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 = lcplcfor 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 commonlystill 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.

http://www-structure.llnl.gov/cd/cdtutorial.htm

CD is used for proteins because of the chiralnature 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 acidsChromophores PHE, TRP, TYR -

When an aromatic residue is held rigidly in space, its environment is asymmetric, and it will exhibit circular dichroism.

Amide bondIn secondary structure conformations, the backbone and

the amide bond chromophores are arranged in regular, organized, asymmetric patterns.

Near-UV CD spectroscopy is dominated by Phe, Tyr, Trp and disulfides

Near UV CD (250 - 350 nm)

The amide group is the most abundant CD chromophore in proteins.

* transition ~ 190 nmn* transition ~220 nm

Far UV CD (180 - 250 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.htmlA 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