For Circular dichroism studies, we know that if the negative elipticity at 208 and 222 are more then it would be alpha helical protein. but if it is more at 208nm or more at 222 nm, what does it mean? does it relate to extent of helical content?
Dear Rakesh,
the ellipticity at 208 and 222 or to be more exact the ratio 222/208 nm tells you whether you have an isolated helix or several helices that form a coiled coil. It is said single helices have a ratio of 0.9 while coiled coils have a ratio of greater than 1.10. However these numbers are not exact and depending on where you look you will find different numbers.
Here are some links for you to read more about this:
https://en.wikipedia.org/wiki/Coiled_coil
http://www.ucdenver.edu/academics/colleges/medicalschool/programs/biomol/strucbiocores/biophysics/instruments/Circular%20Dichroism%20Optical%20Rotary%20Dispersion/Pages/default.aspx
Best regards, Alex
Dear Rakesh,
the ellipticity at 208 and 222 or to be more exact the ratio 222/208 nm tells you whether you have an isolated helix or several helices that form a coiled coil. It is said single helices have a ratio of 0.9 while coiled coils have a ratio of greater than 1.10. However these numbers are not exact and depending on where you look you will find different numbers.
Here are some links for you to read more about this:
https://en.wikipedia.org/wiki/Coiled_coil
http://www.ucdenver.edu/academics/colleges/medicalschool/programs/biomol/strucbiocores/biophysics/instruments/Circular%20Dichroism%20Optical%20Rotary%20Dispersion/Pages/default.aspx
Best regards, Alex
Thanks Alex, What I observed is -for a wild type protein (c-terminus is known to have 5 helix bunddle) ratio is more than 1 but with a single amino acid change the ratio has shifted to 0.9 at all temperatures. for another mutant with single amino acid change the ratio was more than 1 at 20C but upon 40C onwards it was again less than one, is this some conformational change or simply unwinding of coil.
The DOSY NMR spectrum at 20C vs 40C would distinguish these possibilities. A conformational change would result in a small change in the diffusion constant while dissociation of a coiled coil assembly would give a much larger change. Since this is a 1D experiment that does not require high resolution it can be performed on large proteins.
More generally, a 222/208 ratio higher than 1.1 may indicate a coiled coil state[4]
A 222/208 ratio distinctly smaller than unity can be evidence of a 310 helix (not likely in this case) [1]. This conformation is relatively unstable and rarely seen in crystal structures, but short stretches are relatively common in NMR structures of peptides and proteins. It can be more positively identified by its particular NOE pattern in NMR ( dαN(i,i+2) NOEs and absence of dαN(i,i+4) NOEs
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