Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs

Abstract

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the tripleresonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13 C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, C α , C β , C′) resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

“…nanodiscs and its resonance assignment is described in Kooijman et al [37] Overall, we assigned 156 backbone amide groups (62 %) and 95 methyl groups (59 %o fA la, Ile(d1), Leu(d1/2), Va l(g1/2)). Additionally we were able to identify the amino acid type of all methyl groups using HMCM-CG/ CB/CA type experiments.…”

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

“…Duplication of many peaks was observed in [ 15 [37] We noticed that doubling in our initial analysis [37] and attribute this peak doubling to the darkadaptation of bR, which results in am ixture of all-trans,15anti and 13-cis,15-syn isomers of retinal. [21] Theisomer ratio of all-trans,15-anti:13-cis,15-syn has been determined previously as 1:1.6 by HPLC for retinal extracted from bR, [23] or 1:1.5 by solid-state NMR [21] similar to the 1:1.6 and 1:2.0 ratio measured by us from the average intensity ratio of corresponding peaks in [ 15 N, 1 H]-TROSY and methyl [ 13 C, 1 H]-TROSY spectra, respectively.S cherrer and co-workers compared the retinal isomer ratio for the bR trimer in purple membranes and the monomer solubilized in Tr iton X-100 [45] and concluded that the isomer ratio is very similar in the two environments.I mportantly,i tw as demonstrated that the bR monomer is capable of pumping protons [46] and hence presents the functional unit for the photocycle.…”

“…Forschungsartikel 21152 www.angewandte.de nanodiscs and its resonance assignment is described in Kooijman et al [37] Overall, we assigned 156 backbone amide groups (62 %) and 95 methyl groups (59 %o fA la, Ile(d1), Leu(d1/2), Va l(g1/2)). Additionally we were able to identify the amino acid type of all methyl groups using HMCM-CG/ CB/CA type experiments.…”

“…Additionally we were able to identify the amino acid type of all methyl groups using HMCM-CG/ CB/CA type experiments. [37,44] Theu nassigned residues are mostly located in the extracellular halves of the helices,t he extracellular loops,a nd the entirety of helix 5. Since we assigned the vast majority of the observable peaks,t his suggests that these parts of the protein undergo intermediate conformational exchange resulting in peaks that are broadened beyond detection (vide infra).…”

“…[31][32][33][34][35][36] To cope with the increased size of the system, we recently developed aprotocol for resonance assignments in the absence of extensive mutagenesis. [37] It uses the program FLYA , [38,39] which combines information from NMR experiments utilizing scalar or dipolar couplings in combination with structural data to form assignments.T he new approach included factors such as proximity to water and lipid molecules,aswell as biochemical information from differential isotope labeling.I nterestingly, many signals from residues in the extracellular half of bR were absent, likely due to intermediate conformational exchange.T wo sets of peaks were present in the spectra arising from the two all-trans,15-anti and 13-cis,15-syn isomeric states of retinal. [37] Herein, we compare spectra in the dark to those recorded upon laser illumination to assign which of the two sets of peaks belong to the light-adapted all-trans,15-anti state.T he availability of methyl group assignments allowed us to measure site-specific methyl group relaxation rates and to compute order parameters from the NMR data.…”