Solution NMR resonance assignment strategies for β‐barrel membrane proteins

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 b-barrel membrane proteins using Opa 60 from Neisseria gonorrhoeae as a model system. Opa 60 is an eight-stranded b-barrel with long extracellular loops (~63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa 60 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 b-barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized b-barrel. The removal of the loop resonances significantly improved the assignment of the Opa 60 b-barrel region (97% of the resonances corresponding to the b-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 b-barrel membrane proteins.

Aβ42 assembles into specific β-barrel pore-forming oligomers in membrane-mimicking environments

“…3F). Second, another characteristic of β-barrel membrane proteins is that proteases generate polypeptide fragments within the solvent-accessible flexible regions while leaving the β-barrel intact (23). Consistent with this finding, SDS/PAGE analysis of the nonboiled Aβ42 oligomer sample previously incubated with proteinase K revealed a band at 11 kDa (Fig.…”

“…These models were derived by using shorter Aβ sequences, ranging from residues 9 to 42 or from 17 to 42, and assuming, without any direct 3D structural data, that each Aβ subunit within the β-barrel adopts the same structure as that of Aβ in the fibril. This assumption leads to a β-barrel formed by double β-sheets (33) instead of a single circular β-sheet, as described for transmembrane β-barrel proteins (18,23). The properties of βPFOs Aβ42 are such that they are amenable to studies designed to obtain their 3D atomic structure, thus providing a unique opportunity to obtain a 3D structure of a pore-forming Aβ oligomer.…”

Structure of the Neisserial Outer Membrane Protein Opa60: Loop Flexibility Essential to Receptor Recognition and Bacterial Engulfment

“…The assignment strategy for Opa 60 is published 12 and mapped onto the 15 N, 1 H-TROSY-HSQC in Figure S1 (Supporting Information). Through these strategies, complete nitrogen, hydrogen, Cα, Cβ, and CO resonances were assigned for residues 1–14, 29–31, 51–71, 95–97, 109–110, 118–140, 157, 159–178, 190–212, 231, and 233–238 (Figure 1B) with only Cα, Cβ, and CO resonances for the seven assigned prolines and a lack of Cβ assignment for two additional resonances (Y71 and I97).…”

Post-expression strategies for structural investigations of membrane proteins

“…The removal of these flexible regions simplifies the NMR spectrum by removing intense resonances that have spectral overlap with weaker resonances from the folded membrane region. Limited proteolysis has proven to be an effective method for assigning β-barrel membrane protein resonances [9]. The β-barrel membrane proteins are highly stable once folded in detergent as indicated by their resistance to unfolding in SDS (even boiled) and, thus, are likely more amenable to this approach than α- helical membrane proteins.…”

“…As a result, limited proteolysis of β-barrel membrane proteins in a membrane mimic with a protease may not result in denaturation. For Opa 60 [10], Opa 50 , and OprH [11], the resulting five fragments after trypsin proteolysis maintained the β-barrel fold and the corresponding resonances were nearly superimposable with that of the untreated proteins (Figure 2B) [9]. The ability to conduct the assignments on the proteolysed sample and map the assignment on to the full length was essential to determining the structure of the Opa 60 β-barrel [9].…”