Recently, I performed CD spectra for completely beta sheet containing protein which is in buffer solution 25mM sodium phosphate (pH 7.5). I scan the spectra from 190nm-260nm spectral range where I got minima at 207nm and maxima at 230 nm instead of minima at 218nm and maxima at 195nm respectively.
Hi Priyanka,
What is the evidence that your protein is "completely beta sheet"? Few if any proteins are 10% beta sheet. Beta-rich globular proteins rarely exceed 50% sheet.
Manavalan and Johnson (Nature 305, 831-832 (1983)) observed that beta-rich proteins fall into two classes with respect to their CD spectra: one class has a classical beta-sheet spectrum (negative band at 218 nm, positive band at 195 nm); the other class has a CD spectrum like that of unordered proteins (negative band near 200 nm). Yang and coworkers (Anal. Biochem.200, 359-364 (1992)) designated these two classes as beta-I and beta-II, respectively. They also showed how beta-II proteins could be distinguished by CD criteria from unordered proteins: beta-II proteins exhibit a sharp thermal transition associated with unfolding and they generally have well-defined near-UV CD spectra associated with their aromatic siode chains. Sreerama and I (Protein Sci. 12, 384-388 (2003)) pointed out that the difference between beta-I and beta-II proteins lies in their poly(Pro)II (PPII)) content. Beta-I proteins have relatively small PPII content relative to their beta content. Beta-II proteins have PPII content comparable to their beta content and the PPII CD spectrum dominates over the beta CD spectrum. If the PPII/beta ratio exceeds ca. 0.4, the proteins exhibits a beta-II CD spectrum, whereas beta-I proteins have PPII/beta < ca. 0.4.
Your observation of a positive band at 230 nm indicates a significant contribution from Tyr and/or Trp side chains. A number of proteins, especially beta-rich proteins, exhibit such bands. This has been discussed in several papers (Woody, Biopolymers 17, 1451-1467 (1978), Eur. Biophys. J. 23, 253-262 (1994); Khan et al., Biochemistry 46, 4565-4579 (2007)).
Robert Woody
Thanku sir for your valuable suggestion. Crystal structure of this protein shows it has immunoglobulin fold having beta pleated sheets connected with loops.this protein is approximately 33kda having 13 phenylalanine, 4 tryptophan and 12 tyrosine. Is having this much aromatic amino acid probable reason for unconventional spectra?
Hi Priyanka,
First, let me correct a typo in my reply from yesterday. In the second sentence, I meant to say that few if any proteins are 100% beta sheet.
Yes, it is possible that the unusual CD spectrum of your protein results from aromatic side chains, but I think it unlikely. It seems much more likely that regions of PPII in the loops connecting beta strands give rise to the negative band at 207 nm. I suggest that you try analyzing the CD spectrum using our CDPro program (Sreerama and Woody, Anal. Biochem. 287, 252-260 (2000)) and a reference set that considers the PPII conformation. You might find a paper by Tetin and Venyaminov (Anal. Biochem. 321, 1830187 (2003)) of interest. They studied 3 IgG Fab fragments with rather different (but all class beta-I), analyzing the secondary structure by CD, and obtained good agreement with the x-ray diffraction structures. I checked the aromatic amino acid content of one of their proteins and found it to be 10.5%, quite comparable to that of your protein (10%). Thus, a high aromatic amino acid content does not preclude accurate CD analysis of secondary structure.
Robert Woody
Hello Priyanka,
In addition to what Prof. Woody discussed and suggested, another idea would be to check near-UV CD spectrum of your protein to make sure that it has unique 3D structure in solution. This will allow you to exclude a possibility that that your protein is random coil. Also, you mentioned that the crystal structure of your protein is known. Did you try to look for the presence of "aromatic clusters" in this structure (i.e,, regions in structure that have several aromatic residues in close proximity to each other). My understanding is the presence of such aromatic clusters in rigid environment may cause unusual shape of beta-structural proteins.
Vladimir
Thanku Dr. Woody and Dr. Vladimir I will take your suggestion and I will get back to you soon with probable solution.
Thanks priyanka
Does it have any enzimatic activity that you can measure? You can try also DLS just to be sure. Could it be that your expression and purification protocol gives you unfolded protein?
Dear Priyanka Chaurasia ,
I am not sure whether it is of any relevance to you after so long, but I stumbled on the following paper which might shed some light on your question:
Article Accurate secondary structure prediction and fold recognition...
They present the use of a novel algorithm, β-structure selection (BeStSel), which reliably distinguishes parallel from antiparallel β-sheets by CD spectroscopy:
http://bestsel.elte.hu/index.php
To me it seems that the spectrum you depicted might resembles (somewhat) a protein that contains a so-called antiparallel β-sheet, right hand twisted (Figure 3B).
I personally have not seen this type of spectra that often but according to the referred paper it might be a possible explanation.
Hope this is of any help to anyone encountering something like this.
PS. For more accurate ideas about a 'true' random coil reference spectrum see
Chapter Characterization of helical structures in gelatin networks a...
Chapter Characterization of helical junction zones in gelatin networ...
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