Optimization of experimental conditions for structural studies of the membrane surface complex between the small GTPase Arf1 and the ArfGAP ASAP1 by paramagnetic relaxation enhancement NMR.

Abstract

Stimulated GTP hydrolysis of the small, membrane bound GTPase, Arf1, by the Arf GTPase activating protein (ArfGAP), ASAP1, participates in the regulation of actin-dependent membrane traffic and organelle structure. We previously showed that a functional complex between Arf1 (∼ 22 kDa) and the core catalytic domain of ASAP1—referred to as ASAP1-PZA (∼ 44 kDa) can be built at the membrane surface of a nanodisc (∼ 90 KDa). For a complex of that size, slow tumbling in solution and spectral crowding can hinder the collection of clear, high-quality NMR spectra. This study aims at optimizing sample preparation and experimental conditions to measure distances between MTSL- tagged Arf1 and isotopically labelled ASAP1-PZA at the membrane surface by paramagnetic relaxation enhancement (PRE) NMR. To minimize the size of the membrane bound complex, we developed conditions to obtain a stable, single Arf1:ASAP1 PZA complex per nanodisc and monitored results by analysis of protein band intensities on SDS-PAGE and SEC chromatography. Influence of temperature on protein stability and spectrum quality was monitored by recording Methyl-TROSY HMQC spectra of U-2H, 15N, ILV 13C methyl labeled Arf1. We show that optimized sample preparation and the use of ILV 13C segmentally methyl labeled PZA allow the collection of distance restraints between Arf1 and the individually labelled PH or ZA domain of ASAP1-PZA at the membrane surface.