molecules-18-07103.pdf (1.45 MB)
0/0

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

Download (1.45 MB)
journal contribution
posted on 18.03.2014 by Tanmay S. Chavan, Sherwin Abraham, Vadim Gaponenko
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.

Funding

American Cancer Society Grant RGS-09-057-01-GMC and the National Cancer Institute Grant R01 CA135341 to Vadim Gaponenko.

History

Publisher Statement

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Publisher

MDPI

Language

en_US

issn

1420-3049

Issue date

01/06/2013

Exports

Categories

Exports