Preparation of H-Ras GTPase conjugated to lipid nanodiscs for NMR spectroscopy

Abstract

Ras GTPase is a peripheral membrane protein central to cellular signaling of growth and proliferation. Membrane attachment is critical for a range of Ras activities, therefore, ability to make faithful in-vitro samples of a mem-brane-bound Ras for detailed biophysical studies is a highly desirable goal. In this manuscript, we are describing preparation of a large-scale sample of isotopically labeled H-Ras conjugated to lipid nanodiscs. We demonstrate that the Ras-nanodisc sample is fairly stable to allow for a range of Nuclear Magnetic Resonance (NMR) and other biophysical measurements. The need to achieve a homogeneous protein-nanodisc ratio is also emphasized.