Revision history [back]
click to hide/show revision 1
initial version

posted Dec 03 '11 at 12:55

sekhar%20Talluri's gravatar image

sekhar Talluri
621

Your description of the data, although not entirely clear, appears to suggest a phenyl-alanine rather than a tyrosine. You should check your assignments first. A triple quantum filter will remove the cross-peak if it involves HD-HE of tyrosine, but will not do so if it is from phenyl-alanine. Therefore, a triple quantum filtered 2D COSY should distinguish between these two possibilities. You cannot rule out the possibility that the amino acid sequence of the protein has been determined incorrectly - I know of a case where NMR data was used in the course of protein structure determination by Prof. Wagner to correct a mistake in the amino acid sequence of the protein. However, the (very unlikely) possibility of a Tyrosine OH cannot be ruled out inspite of the unusual chemical shift and the presence of cross peaks in D2O. If the Tyrosine OH is buried and hydrogen bonded, then mild heating in D2O may not lead to exchange with solvent D2O. If your protein can be completely unfolded by heating in D2O, and then renatured by cooling, then the cross peak should dissappear from D2O spectrum if the cross peak involves a J coupling between Tyrosine OH-HE. Note: The thermal denaturation and renaturation should be carried out in a dilute solution of the protein in D2O, to prevent aggregation.
click to hide/show revision 2
No.1 Revision

posted Dec 03 '11 at 12:57

sekhar%20Talluri's gravatar image

sekhar Talluri
621

Your description of the data, although not entirely clear, appears to suggest a phenyl-alanine rather than a tyrosine. You should check your assignments first.

A triple quantum filter will remove the cross-peak if it involves HD-HE of tyrosine, but will not do so if it is from phenyl-alanine. Therefore, a triple quantum filtered 2D COSY should distinguish between these two possibilities.

You cannot rule out the possibility that the amino acid sequence of the protein has been determined incorrectly - I know of a case where NMR data was used in the course of protein structure determination by Prof. Wagner to correct a mistake in the amino acid sequence of the protein.

However, the (very unlikely) possibility of a Tyrosine OH cannot be ruled out inspite of the unusual chemical shift and the presence of cross peaks in D2O. If the Tyrosine OH is buried and hydrogen bonded, then mild heating in D2O may not lead to exchange with solvent D2O. If your protein can be completely unfolded by heating in D2O, and then renatured by cooling, then the cross peak should dissappear from D2O COSY spectrum if the cross peak involves a J coupling between Tyrosine OH-HE.

Note: The thermal denaturation and renaturation should be carried out in a dilute solution of the protein in D2O, to prevent aggregation.

powered by CNPROG