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Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs
- Source :
- Journal of Biomolecular Nmr, Journal of Biomolecular NMR, 74 (1)
- Publication Year :
- 2019
-
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 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. Electronic supplementary material The online version of this article (10.1007/s10858-019-00289-7) contains supplementary material, which is available to authorized users.
- Subjects :
- 10120 Department of Chemistry
0301 basic medicine
Models, Molecular
1303 Biochemistry
Stereochemistry
1607 Spectroscopy
membrane proteins
010402 general chemistry
01 natural sciences
Biochemistry
Peptide Mapping
Article
03 medical and health sciences
Residue (chemistry)
540 Chemistry
Amino Acid Sequence
Resonance assignment
Membrane protein
Solution-state NMR
Nanodisc
Spectroscopy
chemistry.chemical_classification
biology
bacteriorhodopsin
Chemistry
Mutagenesis
Bacteriorhodopsin
Cyana
biology.organism_classification
NMR
3. Good health
0104 chemical sciences
Amino acid
assignment
030104 developmental biology
Bacteriorhodopsins
ddc:540
biology.protein
Nanoparticles
Two-dimensional nuclear magnetic resonance spectroscopy
Algorithms
Subjects
Details
- ISSN :
- 15735001
- Volume :
- 74
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Journal of biomolecular NMR
- Accession number :
- edsair.doi.dedup.....ffe1d06d5c53a9639d5c53931de86b68