Dear Nathaniel Ninyio ,

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”.

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

Unfortunately the deconvolution of CD spectra varies a lot and depends on the method used and some tent to over-estimate the alpha helical content. So an absolute answer based on one method is not THE answer of the analysis of your spectrum. The best you can do is comparing the results of different CD analysis since numerous programs are available (often your CD equipment includes pretty good software as well).:

Dichrocalc:

http://comp.chem.nottingham.ac.uk/dichrocalc/

Dichroweb:

http://dichroweb.cryst.bbk.ac.uk/html/process.shtml

BestSel:

http://bestsel.elte.hu/

K2D3:

http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3//

Dicroprot:

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

There are many more different programs to deconvolute your CD signal in order to extract secondary structure contents (CDPro, CDNN etc.).

For a nice introduction see:

https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/CD%20spectroscopy.pdf

PS. 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

or

JPred

http://www.compbio.dundee.ac.uk/jpred/

Hope this answers your question somewhat. Good luck!

Dear Nathaniel Ninyio

all what Rob Keller said is true: you don't have to trust blindly a single deconvolution provided by a unique program. From my experience, the CONTIN is one of the most robust method for deconvolution.

On the mean time, there is two aspects that have to be kept in mind for CD data analysis:

* it's true that having a high beta content seems to be surprising when the data displays a clear alpha helix profile. But this apparent contradiction can be explained when you consider the profiles of pure beta and random contents. In fact, these two contributions are roughly opposite in sign, which means that equal contributions of beta and random (as it is in your case with 45% beta sheets and 39% random) almost cancel each other out in the global CD signal.

* secondly, the deconvolution programs are highly sensitive to the concentration of your protein. Indeed 5 to 10 % difference in the concentration may result in several tens of % change of the secondary structure content estimate. Here we are back to a common problem andlimitation in CD that is an accurate estimate of the protein concentration. For sure, the Bradford analysis is not sufficient. Absorption at 280 nm might be appropriate, provided that aromatic residues (ans specially tryptophans) are present in the primary sequence of your polypeptide. But you have definitively to pay a special attention to this critical point. Therefore, you also have to slightly change the concentration of your protein according to the uncertainty of you protein concentration method in order to evaluate the impact on the secondary structure content estimates.

Hope this will help you.

Best

Yves

Accurate CD estimation of secondary structure levels is fundamentally dependent upon accurate determination of the protein concentration. Likely the method of protein estimation is off. Spuriously high level of helical content is consistent with underestimation of sample protein concentration.

Contamination with highly helical protein would also do this.

@RobKeller, @YvesNomine and @EricTurk, thank you for your helpful responses.