TY  - JOUR
A1  - Kooijman, Laurens
A1  - Ansorge, Philipp
A1  - Schuster, Matthias
A1  - Baumann, Christian
A1  - Löhr, Frank
A1  - Jurt, Simon
A1  - Güntert, Peter
A1  - Zerbe, Oliver
T1  - Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs
T2  - Journal of biomolecular NMR
N2  - 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 triple-resonance 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 13C 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, Cα, Cβ, 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.
KW  - Resonance assignment
KW  - Membrane protein
KW  - Solution-state NMR
KW  - Nanodisc
Y1  - 2019
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/53552
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-535525
SN  - 1573-5001
N1  - Open Access: This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
VL  - 74
IS  - 1
SP  - 45
EP  - 60
PB  - Springer Science + Business Media B.V
CY  - Dordrecht [u. a.]
ER  -