Molecules 2013, 18(6), 7103-7119; doi:10.3390/molecules18067103
Article

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

Received: 20 May 2013; in revised form: 13 June 2013 / Accepted: 14 June 2013 / Published: 18 June 2013
(This article belongs to the Special Issue NMR of Proteins and Small Biomolecules)
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract: 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 13C-methylation to study protein interactions at low micromolar concentrations. Methyl groups in dimethyl lysines are degenerate with one 13CH3 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 13C-reductive methylation of lysines to enhance the sensitivity of conventional NMR methods for studying protein interactions at low concentrations.
Keywords: K-Ras; calmodulin; reductive methylation; hypervariable region; NMR
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MDPI and ACS Style

Chavan, T.S.; Abraham, S.; Gaponenko, V. Application of Reductive 13C-Methylation of Lysines to Enhance the Sensitivity of Conventional NMR Methods. Molecules 2013, 18, 7103-7119.

AMA Style

Chavan TS, Abraham S, Gaponenko V. Application of Reductive 13C-Methylation of Lysines to Enhance the Sensitivity of Conventional NMR Methods. Molecules. 2013; 18(6):7103-7119.

Chicago/Turabian Style

Chavan, Tanmay S.; Abraham, Sherwin; Gaponenko, Vadim. 2013. "Application of Reductive 13C-Methylation of Lysines to Enhance the Sensitivity of Conventional NMR Methods." Molecules 18, no. 6: 7103-7119.

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