It is important because in order to compare to different proteins or when you want to use software for the determination of the secondary structure content, you have to convert the CD signal (in mdeg) to ThetaMRW (= mean Ellipticity per amino acid residue). One important factor that goes into this conversion is the protein concentration. Thus protein concentration is a critial factor when you do CD. Check out this paper from Kelly & Price: http://www.ncbi.nlm.nih.gov/pubmed/12369905
Cheers
It is important because in order to compare to different proteins or when you want to use software for the determination of the secondary structure content, you have to convert the CD signal (in mdeg) to ThetaMRW (= mean Ellipticity per amino acid residue). One important factor that goes into this conversion is the protein concentration. Thus protein concentration is a critial factor when you do CD. Check out this paper from Kelly & Price: http://www.ncbi.nlm.nih.gov/pubmed/12369905
Cheers
Alexander resumed perfectly.
I will just add one suggestion : when you normalize your data to convert them in mdeg or Theta, try to introduce some variation in your determined protein concentration and compare the deconvolution obtained with these “approximate” converted data’s to yours normal one. You can observe that a 5% variation in the protein concentration can introduce until 20% variation in the secondary structure estimated content.
So, I will add this remark: when you perform protein quantification, are you totally sure of your estimation? We observed for example that quantification for certain proteins in certain buffers performed on a NanoDrop can give up to 30% variation in protein content compared with that we obtain with the same preparation on a classical double beam spectrophotometer.
As mentioned by me in a former answer e.g. in these classical papers you can find some background on CD analysis of protein secondary structure and related Software.
*Sreerama, N., and Woody, R.W. 2000 Anal Biochem 287, 252
*N.J. Greenfield, Nat Protoc. 2006 ; 1(6): 2876
*Also JASCO company comes with a tool ("Secondary structure estimation"), where most of latest software is implemented.
As stated in a former answer, protein concentration and the pathlength of your cuvette are crucial input parameters in all these softwares. Very importantly, protein concentration is related to the concentration of the peptide units (nearly eq. to amino acids: protein with 100 amino acids has 99 peptide units) and not to the concentration of the whole protein (much higher molar mass) ! As an approximation for the molar mass of amino acids one can work with around 100 g/mol (exactly, you can sum up all molar masses of the amino acids of your protein and divide by the total number of amino acids of your protein). This is done so, since these softwares use CD spectra of many other proteins with different molar masses but known secondary structure, to quantify the secondary structure of your protein. [less]
Hello Dr. Alexander, Dr. Bruno and Dr. Martin,
Thanks for your answers.
I would also like to know how can we detect the effect of mutation in protein in case there is no aromatic amino acids and we have to use far UV? I want to know the principle behind it because in any case no. of peptide bonds will remain the same.
I understand that the mutation will lead to a change in secondary structure but again the no. of peptide bonds will remain the same, I guess.
Jitendra
Hello Jitendra,
Your question includes the answer: there is absolutely no influence of aromatic residues (absent or present) on far-UV CD as only peptide bond is absorbing in this region. So, as you are looking at the overall secondary structure of your protein, any change that you can measure between normalized curve of your WT and mutant clones can be related to the presence of the mutation.
hello Dr. Bruno..Thanks for replying....As your rightly mentioned any change that can bemeasured between normalized curve of your WT and mutant clones can be related to the presence of the mutation; however I would also like to know if there is direct relation between peptide bond and mutation?
thanks
There could be a relationship between the peptide bond and the mutation, but only if the secondary structure is affected by the mutation. This will change the peptide bond orientation and potentially change the CD signal. Be warned, though, a fair percentage of your peptide bonds have to be dramatically different in order to see a change in Far-UV region of CD. CD is a low resolution technique in this regard. You are better off trying H/D exchange MS.
Thanks Dr. Lin,
Thats what I was trying to know through my second question. I got to know the same thing from one of my Professors today.
regards
Jitendra
Hello Jitendra,
since your mutant lack aromatic amino acid, l would recommend ninhydrin assay in order to determine your protein concentration.
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