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Protein Sci. 2013 Aug;22(8):1133-40. doi: 10.1002/pro.2291. Epub 2013 Jun 27.

Solution NMR resonance assignment strategies for β-barrel membrane proteins.

Author information

1
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.

Abstract

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β-barrel membrane proteins using Opa60 from Neisseria gonorrhoeae as a model system. Opa60 is an eight-stranded β-barrel with long extracellular loops (∼63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa60 in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β-barrel. The removal of the loop resonances significantly improved the assignment of the Opa60 β-barrel region (97% of the resonances corresponding to the β-barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full-length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β-barrel membrane proteins.

KEYWORDS:

NMR; assignments; outer membrane protein; β-barrel membrane protein

PMID:
23754333
PMCID:
PMC3832050
DOI:
10.1002/pro.2291
[Indexed for MEDLINE]
Free PMC Article

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