help with circular dichroism

Robert Cerpa cerpa at cgl.ucsf.edu
Fri May 31 00:56:29 EST 1996


In article <1996May27.171533.13730 at cs.mun.ca> alvin at cs.mun.ca (Alvin Dobbin) writes:
>To anyone who can help,
>
>I have been using circular dichroism to study the thermal
>stability of an à-helical protein. When the unfolding data is
>plotted as: (1) relative ellipticity at 222 nm versus T and 
>(2) relative ellipticity at 208 nm versus T, the profiles are
>somewhat similar but not identical. What is the reason for the
>difference ? What electronic transitions do the minima at 208 nm
>and 222 nm correspond to ?
>
>I am using a Jasco- J-500 A. The records are from sixteen repeats
>(260-200 nm).
>
>If you can provide any information you feel may be helpful it
>would be greatly appreciated.
>
>Thank you very much,
>Alvin Dobbin.

hi Alvin.  One possible reason for your discrepancy may be that
aromatic residues could be distorting the intensities of the minima,
most likely the 222nm n-to-pi-star transition.  If you take a look at
Chakrabartty, A. et al., Biochem 32:5560 (1993) (a paper from Professor
Baldwin's lab) and Vuilleumier, S. et al., Biochem 32:10303 (1993),
(from Prof. Fersht's lab) you'll see examples of mild to gross
distortions of the CD spectra of various peptides and proteins due to
tyrosines and tryptophans (I don't believe that the papers found a
problem due to phe or his).

The electronic transitions involved are an electron from the
non-bonding orbital on oxygen going to the pi-star molecular orbital of
the amide group (the n-to-pi-star transition at 222nm in the helix) and
an electron going from the pi molecular orbital to the pi-star
molecular orbital (pi-to-pi-star transition at 208nm).

Tyrosine, tryptophan, and the disulfide bond have electronic
transitions in the 220-230nm region (trp and tyr also have bands at
higher wavelength, which are much more useful since they don't overlap
with the peptide bond electronic transitions), and as the papers
above indicate, if the side chain is in an asymmetric environment,
those transitions will have an effect on the far-UV spectrum.

Of course, if you DON'T have aromatic residues in your protein, it's
back to the drawing board!


Robert Cerpa, Ph.D.
Registered Patent Agent (but I used to be a scientist in a former life...)



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