A 1D sensitivity-enhanced 1H spin diffusion experiment for determining membrane protein topology

Abstract

A sensitivity-enhanced 1D 1H spin diffusion experiment, CHH, for determining membrane protein topology is introduced. By transferring the magnetization of the labeled protein 13C to lipid and water protons for detection, the CHH experiment reduces the time of the original 2D 13C-detected experiment by two orders of magnitude. The sensitivity enhancement results from 1H detection and the elimination of the 13C dimension. Consideration of the spin statistics of the membrane sample indicates that the CHH sensitivity depends on the 13C labeling level and the number of protein protons relative to the mobile protons. 5–35% of the theoretical sensitivity was achieved on two extensively 13C labeled proteins. The experimental uncertainties arise from incomplete suppression of the equilibrium 1H magnetization and the magnetization of lipid protons directly bonded to natural-abundance carbons. The technique, demonstrated on colicin Ia channel domain, confirms the presence of a transmembrane domain and the predominance of surface-bound helices.