Investigating the Influences of Lipid Binding on Rhodopsin Activation using Native Mass Spectrometry

Abstract

Methyl-TROSY NMR of specifically 13 C methyl-labeled proteins associated to nanodisc bilayers is a powerful tool to probe the structure of membrane-bound macromolecular complexes. However, protein methyl resonances are obscured by baseline artefacts stemming from the strong signals of the lipid chains. While artefacts can be eliminated by using lipids with perdeuterated acyl chains, this strategy is prohibited when complex lipid mixtures are required for protein function. Here, we compared three approaches to selectively remove the lipid signals, using the ArfGAP ASAP1 PH domain and the small GTPase Arf1 as representative proteins. (I) Spin Label Induced PRE (SLIP). The bilayer was doped with a small fraction of doxyl spin labeled lipids at position 5, 10 or 16 along the acyl chain, or 25-doxyl cholesterol (CNO) to induce paramagnetic line broadening of the lipid resonances. (II) Selective excipient reduction and removal (SIERRA), a 1 H/ 13 C frequency selective filter proposed by Arbogast et al. (III) Lipid-Proton Driven Spin Diffusion (L-PDSD) from the lipid methylene signal to the lipid methyl signal. Addition of 2 mol% of 10-doxyl lipid or CNO attenuated both the lipid methylene and methyl signals by a factor of 5. In combination with L-PDSD, lipids signals were completely removed. Protein methyls were attenuated in a distance dependent fashion with severe broadening for methyl located at the membrane phosphate plane or below. On the contrary, application of a SIERRA filter caused only minor reduction of protein methyl resonances at high field, while attenuating the lipid methyl signal by a factor of 10. The improvement in data quality allowed us to determine the interface between Arf1 and ASAP1 at the membrane surface using solvent PRE and chemical shift perturbations.