Dear Lily

can you share the different profiles?

the change of secondary structure with the concentration it sound stange to me, is it possible that at high concentration peptide tend to aggregate or it contain different amount of some solvent eg DMSO which can affect the conformation?

However in the past i used Dicorpot software package wich

https://dicroprot-prabi.ibcp.fr/

which compare the result of 6 different methods

good luck

Manuele

Dear Lily Lin ,

If I recall correctly the method the Jasco software uses is based on:

Greenfield, N. J., & Fasman, G. D. (1969). Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry, 8(10), 4108-4116.

http://garfield.library.upenn.edu/classics1982/A1982NT91500001.pdf

Like other programs as well:

http://rwjms.rutgers.edu/research/cdf/experimental/other.html

If I also recall correctly the software provided by Jasco is based on the shape of the curve (at least this is one of the options) and not on absolute values (corrected for concentration and so on).

Other methods like the mentioned CDNN are based on other principles and/or reference spectra so a difference in outcome using different methods of deconvolution of your spectra is (unfortunately) often found.

You can compare your result with the so-called 220 nm estimate result, see for example:

https://www.researchgate.net/publication/21844323_Anionic_phospholipids_are_essential_for_a-helix_formation_of_the_signal_peptide_of_prePhoE_upon_interaction_with_phospolipid_vesicles

Circular dichroism is relatively good in comparing (relative) results where for example your peptide is studied at different temperatures, with or without anionic phospholipids and so on. This allows you to say “upon increase of temperature one sees a decrease in helical content”.

To get some ‘grip’ on what to expect in terms of secondary structural elements you might consider to do some secondary structure prediction, like:

SOPMA

https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html

Hope this answers your question somewhat. Good luck!

Dear Lily,

I remember that the molar ellipticity is calculated from CD raw data by simple multiplication and division by several factors, which is equivalent to a scaling procedure. The data is normalized to make it comparable to other spectra. When you use a higher protein concentration, the CD absorption (which is actually a special kind of UV absorption) will rise proportionally to the concentration, and normalizing these data will end up in the same scaled data as you obtain with the lower protein concentration. Such a scaling step does not change the intrinsic characteristic shape of the CD spectrum, and therefore the structural contributions obtained by deconvolution of the data are not expected to change when you use any algorithm calculating an appropriate linear combination of spectra for basic structures (like alpha helix and so on) representing your data best. Of course this is only true if you assume that the change in concentration does not affect the protein structure, but this is usually the case in the very dilute protein solutions commonly used.

Hope this helped!

Best regards,

Joscha

Hi Lily,

The secondary structure won't necessarily change with a change in sample concentration, unless increasing concentrations causes new interactions within the protein, changing the secondary structure.

Additionally, you will need to account for the amino acid residues when performing secondary structure estimations.

I'd be happy to discuss your data further if you'd like.