I recently performed CD analysis for my protein of interest in 50mM sodium phosphate buffer. The curve that I got had a dip at 218 nm and 208 nm with a peak at 195 nm ( Keeping in mind that alpha helix has a dip at 222 nm and 208 and a peak at 193 nm whereas beta sheet has a dip at 218 nm and a peak at 195 nm). What can I infer from this data about the secondary structure of my protein. Is it alpha helix or beta sheet or a mixture of both of them. Also, how can I justify the presence of dips and peaks characteristic for both alpha helix and beta sheet.
Homology modelled structure for this protein shows the presence of maximum alpha helix, then beta sheet and few random coils.
Dear Deekshi,
the spectrum of a pure alpha helix, in water, has the two negative peaks you're talking about. I think that you are dealing with a mixture of both with a prevalence of alpha, but if you could report your CD spectrum here, I think this can help people giving you better answers. For a quantitative distinction of the presence of a mixed alpha-beta structure, the solution lies in the deconvolution of the spectrum (there are many available software, CDNN is a very valuable one, which I think is also downloadable for free) and in a comparison of results with a complementary characterization (crystallographic data, I suggest you to consult Protein Data Bank). If the amount of beta structures is much higher than you expected, it is possible that the presence of beta structures can also be due to phenomena of protein-protein interaction. One of the problems is also the use of phosphate buffer, which has high absorption in the spectral region around 195, compromising the linearity of the instrument response.
Greetings,
Federico
Dear Deekshi,
the spectrum of a pure alpha helix, in water, has the two negative peaks you're talking about. I think that you are dealing with a mixture of both with a prevalence of alpha, but if you could report your CD spectrum here, I think this can help people giving you better answers. For a quantitative distinction of the presence of a mixed alpha-beta structure, the solution lies in the deconvolution of the spectrum (there are many available software, CDNN is a very valuable one, which I think is also downloadable for free) and in a comparison of results with a complementary characterization (crystallographic data, I suggest you to consult Protein Data Bank). If the amount of beta structures is much higher than you expected, it is possible that the presence of beta structures can also be due to phenomena of protein-protein interaction. One of the problems is also the use of phosphate buffer, which has high absorption in the spectral region around 195, compromising the linearity of the instrument response.
Greetings,
Federico
With 50 mM phosphate your signal below 200nm is most likely not usable. What is protein concentration? What pathlength did you use? Dynode voltage?
For CD spectra deconvolution you can also check Dichroweb http://dichroweb.cryst.bbk.ac.uk/html/process.shtml
try BestSel, easy and useful (http://bestsel.elte.hu/)
and K2D3, fast (http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3//)
do not consider bands with fixed values: both n-pi and pi-pi bands can move depending on several properties. For instance, the n-pi band moves from 216 to 223 nm from b-sheets to a-helix because this band is polarity-sensitive. The pi-pi band is unique (negative for random while positive for b-sheet) or splitted for a-helix (193 and 208 nm), ... and consider also that side chains give dichroism signal in the far-UV (not only in the near-UV). Check the excellent book: CD and the conformational analysis of biomolecules by GD Fasman for more info.
Dear Deekshi,
There is many different programs to deconvoluate your CD signal in order to extract secondary structure contents (Dichroweb, BestSel, CDPro, ...).
You also have to keep in mind that, the lower the signal, the worst the signal-to-noise ratio. At some points, it could also be usefull to repeat deconvolution with a reduced wavelength range (excluding data at lower wavelength) in order to check the quality of the fits.
Sincerely
Yves
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