A Novel Tool for Probing Membrane Protein Structure: Solid-State NMR with Proton Spin Diffusion and X-Nucleus Detection

Abstract

We present a new solid-state NMR approach, based on 1H spin diffusion with X-nucleus (15N, 13C, 31P) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman−Shen 1H spin-diffusion experiment with X-nucleus detection to proteins in hydrated lipid bilayers (>25% water by weight) in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H2O molecules, which can be selected efficiently by the 1H T2 filter in the Goldman−Shen sequence. The transfer of 1H magnetization from mobile H2O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide 15N signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spin-diffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the 31P in the lipid phosphate head groups and the 13C nuclei in the lipid acyl chains as distance markers for the spin diffusion.