Paramagnetic doping of a 7TM membrane protein in lipid bilayers by Gd3+-complexes for solid-state NMR spectroscopy

A considerable limitation of NMR spectroscopy is its inherent low sensitivity. Approximately 90 % of the measuring time is used by the spin system to return to its Boltzmann equilibrium after excitation, which is determined by ¹H-T₁ in cross-polarized solid-state NMR experiments. It has been shown that sample doping by paramagnetic relaxation agents such as Cu²⁺-EDTA accelerates this process considerably resulting in enhanced sensitivity. Here, we extend this concept to Gd³⁺-complexes. Their effect on ¹H-T₁ has been assessed on the membrane protein proteorhodopsin, a 7TM light-driven proton pump. A comparison between Gd³⁺-DOTA, Gd³⁺-TTAHA, covalently attached Cu²⁺-EDTA-tags and Cu²⁺-EDTA reveals a 3.2-, 2.6-, 2.4- and 2-fold improved signal-to-noise ratio per unit time due to longitudinal paramagnetic relaxation enhancement. Furthermore, Gd³⁺-DOTA shows a remarkably high relaxivity, which is 77-times higher than that of Cu²⁺-EDTA. Therefore, an order of magnitude lower dopant concentration can be used. In addition, no line-broadening effects or peak shifts have been observed on proteorhodopsin in the presence of Gd³⁺-DOTA. These favourable properties make it very useful for solid-state NMR experiments on membrane proteins.