I was measuring lyophylized bovine thrombin (purchased form Sigma Aldrich, http://www.sigmaaldrich.com/catalog/product/sigma/t4648 : composition | Protein, 40-60%, Lyophilized powder containing sodium chloride and Tris-HCl, pH 7.0.) Raman spectrum and found out a strange (as I see it) thing. According to PDB X-ray structure (for example, 1uvs and 1ppb(human)) the 4 disulfide bonds in thrombin have ggg (cys168-cys182 and cys122-cys1) and tgg (cys42-cys58 and cys191-cys220) conformations. But in the measured Raman spectrum I could define peaks on all three frequences, commonly assosiated with different conformations of SS-bonds (~510, 525 & 540(!) cm-1), what indicates the presence of tgt conformation in lyophylized state. Is it possible, that there is such differ between X-ray structure and lyophylized powder?
P.S. I checked, this is not the contribution of Tris-HCl buffer.
Well the data may speak for itself here. This is an interesting observation that may be providing novel insights. I am not aware of data examining this question. In principle, the crystal structure may be only one of the equilibrium conformations from a solution ensemble. Certainly we see from small angle x-ray scattering that some structures in the solution ensemble are not seen in existing structural structures. It might be good to do a study with a few more examples to test how general might be the implications of your new data.
Have you tried powder diffraction rather than crystal? I realise that large proteins may not be ideal for powder, but if it is possible it would be worth a try. You should try measuring the sample in as similar a state as possible between the two techniques. If the powder agrees with the Raman not the crystal X-ray, then you have further supporting evidence that there is a change. As John points out data always speaks for itself, as long as you can rule out sources of error.
Thank you for your attention to my question, mr. Tainer and mr. Beattie. The main idea was to ask if this is a common situation, or something nearly impossible. From general point of view, that is quite normal due to relaxation of the crystal structure and interactions with solvent. But as I found out from literature, quite often it is belived, that there is no structural changes.
Dear James, I am quite amazed with your answer, as I was absolutely sure, that the only possibility for measuring X-ray structure for such complicated molecules, as proteins, is measuring in monocrystals. Is there a possibility to study powder structure?
Depends on the size of the protein, I have no idea of the size of yours. It is likely only possible for relatively small peptides. In answer to your question, it is entirely reasonable that the conformations could change between crystal and powder since the treatment of the sample must have been different to obtain two different physical forms of the sample. If they have been treated differently, it is perfectly reasonable that there is a real difference between the two samples. A further complication is that Raman is much more sensitive to subtle imperfections than X-ray crystallography. I've saw 25 PC components returned from Raman spectra recorded from an apparently pure sample of a solid sample that is not considered polymorphic by crystallographers.
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