Ronald Smulders wrote:
>> Hi CD experts
>> I'm working on a typical beta-sheet protein. So the CD spectrum displays a
> minimum at about 218 nm and the curve crosses the x-axis at about 200 nm.
> Now, when we analyze the protein after a period of heat exposure we see
> an increase of the amplitude: so the spectrum does not shift but only the
> ellipticity increases. Does anyone know how to interprete this result? Does
> this result indicate that the amount of random coil increases upon heating?
>> Thanks in advance for your comments
>> Ronald Smulders, The University of Nijmegen
No claim here to be a CD 'expert', but I can think of two explanations:
1) one possible trivial explanation is that the protein concentration
increased when you heated the sample due to evaporation of water.
Obviously whether this is the true explanation depends on exactly how
you did the experiment, but this can happen more easily than you might
think.
2) The more likely explanation is that you are forming denatured forms
of the protein that contain more beta sheet. It is quite common for
heat-denatured proteins to form very stable intermolecular beta sheets,
so beta sheet content can rise even though you are denaturing the
protein. Usually when this happens the light scattering goes up as
aggregates are formed, and this may lead to higher photomultiplier
voltages to compensate for the loss of light due to scattering. Many CD
instruments save a record of the PM voltage during the scan, so you
might look at that. If indeed you are forming denatured states, you
should see significant changes in the tertiary structure peaks in the
near-uv CD spectrum.
If you could possibly get someone to do FTIR spectra on this protein
that would help a lot. FTIR is generally much better at picking up beta
structure, and can often distinguish parallel from anti-parallel. FTIR
is complementary to CD, which is very good at alpha helix but not so
good at beta sheet.
' Hope this helps. Good luck.
John Philo, Protein Chemistry
Amgen Inc., Thousand Oaks, CA
jphilo at amgen.com
*** Disclaimer: These are the opinions of the poster not Amgen Inc.***
