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

Abstract

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 (1)H-T1 in cross-polarized solid-state NMR experiments. It has been shown that sample doping by paramagnetic relaxation agents such as Cu(2+)-EDTA accelerates this process considerably resulting in enhanced sensitivity. Here, we extend this concept to Gd(3+)-complexes. Their effect on (1)H-T1 has been assessed on the membrane protein proteorhodopsin, a 7TM light-driven proton pump. A comparison between Gd(3+)-DOTA, Gd(3+)-TTAHA, covalently attached Cu(2+)-EDTA-tags and Cu(2+)-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, Gd(3+)-DOTA shows a remarkably high relaxivity, which is 77-times higher than that of Cu(2+)-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 Gd(3+)-DOTA. These favourable properties make it very useful for solid-state NMR experiments on membrane proteins.

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

“…Therefore, this set of experimentally measured relaxation data infers that the DNP agent, AMUPol, could be dispersed relatively homogeneously in the NMR sample. If needed, it is possible to use paramagnetic agents that are bound to the membrane as demonstrated elsewhere [48,65-69]. …”

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…This is made easier by fast spinning that mitigates the need for heteronuclear decoupling, thus lowering the power deposition on the sample and on the coil [106]. Such approach has been successfully used to different kinds of samples, including microcrystalline proteins [107], membrane proteins [108,109] and protein aggregates [110].…”

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

“…They can reduce the longitudinal relaxation times so that data can be acquired much more quickly (Inubushi and Becker 1983; Parthasarathy et al 2013; Ullrich et al 2014; Ward et al 2014), and they can weakly align proteins in solution (Prestegard et al 2000; Tolman et al 1995), providing an alternative to conventional alignment media for the measurement of residual dipolar couplings (RDCs).…”