Detection Of Two Methylated Proteins Based On Nuclear Magnetic Resonance

Methylation is one of the most important post modifications for protein,it plays an important role in modulating cellular and biological processes,including transcriptional regulation,RNA processing,metabolism and signal transduction.NMR is a technique enabling structure analysis of proteins in living cells,however,NMR based protein methylation detection usually requires selective isotope labeling,which is usually difficult to achieve.Herein,maximum-quantum correlation spectroscopy(MAXY)has been introduced to detected methylated proteins in cell lysate.The maximum quantum of CH3 that can be excited are higher than other CHn(n=1-2)groups,therefore,it can directly detect methyl groups of protein.The chemical shifts of the modified methyl groups are usually far away from that of the other side-chain methyl groups,therefore,the method can be used to directly monitor the methylation process of protein in complex bio-system,such as cell lysate.Methyl groups are usually located in the surface of protein,they are sensitive to conformation change or ligand binding of the protein,by selective detecting the methyl groups,the method can also be a sensitive probe for conformation transition of protein even in natural isotopic abundance.To verify the validation of the method,the methylation of H3 and the conformation transition of cytochrome c have been detected,the results are as below:1.The methylation of H3Histone are primary block of nucleosome,which usually undergoes multiple types of post-modifications and impacts numerous nuclear functions.Methylation is one of the typical modifications of histone,which have variant epigenetic regulatory functions.NMR has already been used to monitor the methylation process of H3 peptide in cell lysate,however,the need of selective 15N and 13C labeled lysine made it difficult to be achieved.At present,MAXY has been applied to monitor the methylation of H3.The MAXY of different types of methylated H3 shows that the resonance of the methylated CH3 are usually locate at the downfield region of CH resonances,and the chemical shift of mono-.bi-,and tri-methyl groups are different from each other,therefore,the method can directly monitor methylation of proteins without any other background aliphatic resonances.The method has also been used to monitor the methylation process of H3 in solution and cell lysate,respectively.It is found that the methylation rate and the methylation ratio of H3 in cell lysate are much different from that in solution,indicating that in cell analysis of the methylation process is necessary.In summary,all the results mentioned above indicate that MAXY is a valid method that can monitor the methylation process of proteins without isotopic labelling,and in living cell.2.Conformation change of cytochrome cCytochrome c is an important multifunctional protein,which plays an important role in respiratory chain and cell apoptosis.The conformational changes of cytochrome c are closely related to its functions.Characterization of the conformational changes of cytochrome c is essential to clarify the molecular mechanism of its functions.NMR is an important tool for characterizing protein conformation in near-in-situ environment,but 13C/15N isotopes are usually required.However,the Cytochrome c is difficult to be labeled with its typical modification(K72Me3).In this paper,we attempt to use 1H-13C MAXY NMR method to characterize the conformational changes of naturally purified wild-type cytochrome c.The 1H-13C MAXY NMR method was used to obtain the methyl spectrum of naturally purified wild-type cytochrome c with good dispersion of methyl signal,effectively filtering out methylene,methylene and other signals,and reduced the overlap of spectrum peaks,so the signal recognition is very easy.Through mutant and isotope labeling,we obtained the assignments of methyl groups of mutant cytochrome c,and then obtained the assignments of methyl groups of wild-type cytochrome c by comparing the methyl signal of the mutant with wild-type cytochrome c.Thereby,the conformational change observation in the redox process of the naturally purified wild-type cytochrome c is achieved.The results showed that the conformation of oxidized and reduced wild-type cytochrome c was obviously different.