Application of Reductive C-13-Methylation of Lysines to Enhance the Sensitivity of Conventional NMR Methods

作者：Chavan Tanmay S; Abraham Sherwin; Gaponenko Vadim^*

来源：Molecules, 2013, 18(6): 7103-7119.

DOI：10.3390/molecules18067103

• K-Ras
• calmodulin
• reductive methylation
• hypervariable region
• NMR

NMR is commonly used to investigate macromolecular interactions. However, sensitivity problems hamper its use for studying such interactions at low physiologically relevant concentrations. At high concentrations, proteins or peptides tend to aggregate. In order to overcome this problem, we make use of reductive C-13-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one (CH3)-C-13 signal arising from two carbons and six protons, as compared to one carbon and three protons in aliphatic amino acids. The improved sensitivity allows us to study protein-protein or protein-peptide interactions at very low micromolar concentrations. We demonstrate the utility of this method by studying the interaction between the post-translationally lipidated hypervariable region of a human proto-oncogenic GTPase K-Ras and a calcium sensor protein calmodulin. Calmodulin specifically binds K-Ras and modulates its downstream signaling. This binding specificity is attributed to the unique lipidated hypervariable region of K-Ras. At low micromolar concentrations, the post-translationally modified hypervariable region of K-Ras aggregates and binds calmodulin in a non-specific manner, hence conventional NMR techniques cannot be used for studying this interaction, however, upon reductively methylating the lysines of calmodulin, we detected signals of the lipidated hypervariable region of K-Ras at physiologically relevant nanomolar concentrations. Thus, we utilize C-13-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.

• 出版日期2013-6