Preparative refolding of small monomeric outer membrane proteins

Schwarzer, Tom; Hermann, Maria; Krishnan, Swati; Simmel, Friedrich C.; Castiglione, Kathrin

doi:10.1016/j.pep.2017.01.012

Cited by 10 publications

(13 citation statements)

References 55 publications

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“…2 H, 13 C, 15 N‐labeled AlkL was prepared as previously described ,. The bilayer sample was reconstituted by addition of lipids to a 0.5 lipid to protein weight ratio and removal of detergent by dialysis.…”

Section: Methodsmentioning

confidence: 99%

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Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

et al. 2018

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Determination of the environment surrounding a protein is often key to understanding its function and can also be used to infer the structural properties of the protein. By using proton‐detected solid‐state NMR, we show that reduced spin diffusion within the protein under conditions of fast magic‐angle spinning, high magnetic field, and sample deuteration allows the efficient measurement of site‐specific exposure to mobile water and lipids. We demonstrate this site specificity on two membrane proteins, the human voltage dependent anion channel, and the alkane transporter AlkL from Pseudomonas putida. Transfer from lipids is observed selectively in the membrane spanning region, and an average lipid‐protein transfer rate of 6 s−1 was determined for residues protected from exchange. Transfer within the protein, as tracked in the 15N‐1H 2D plane, was estimated from initial rates and found to be in a similar range of about 8 to 15 s−1 for several resolved residues, explaining the site specificity.

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Section: Methodsmentioning

confidence: 99%

“…2 H, 13 C, 15 N-labeled AlkL was prepared as previously described. [33,34] The bilayer sample was reconstituted by addition of lipids to a 0.5 lipid to protein weight ratio and removal of detergent by dialysis. 2D crystalline 2 H, 13 C, 15 N-labeled hVDAC was prepared using established expression and refolding protocols.…”

Section: Methodsmentioning

confidence: 99%

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

et al. 2018

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“…36 AlkL is involved in transport of hydrophobic molecules and comprises eight TM β-strands as predicted by sequence homology (Figure 4a). U- terms of sensitivity, density of cross peaks and has lower RF requirement, making it particularly suitable for protein structure determination.…”

Section: Toc Graphicsmentioning

confidence: 99%

Selective ¹H–¹H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR

Jain

Lalli

Staněk

et al. 2017

J. Phys. Chem. Lett.

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Very fast magic-angle spinning (MAS > 80 kHz) NMR combined with high-field magnets has enabled the acquisition of proton-detected spectra in fully protonated solid samples with sufficient resolution and sensitivity. One of the primary challenges in structure determination of protein is observing long-range 1H–1H contacts. Here we use band-selective spin-lock pulses to obtain selective 1H–1H contacts (e.g., HN–HN) on the order of 5–6 Å in fully protonated proteins at 111 kHz MAS. This approach is a major advancement in structural characterization of proteins given that magnetization can be selectively transferred between protons that are 5–6 Å apart despite the presence of other protons at shorter distance. The observed contacts are similar to those previously observed only in perdeuterated proteins with selective protonation. Simulations and experiments show the proposed method has performance that is superior to that of the currently used methods. The method is demonstrated on GB1 and a β-barrel membrane protein, AlkL.

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“…Previously, it has been shown that OprG displays a single channel conductance of 500 pS in 1 M KCl (16). More recently, another group showed that the OprG channel has a conductance of 330 pS (28). These values are large and not consistent with a pore through a small monomeric ␤-barrel; they are more typical for oligomeric porins.…”

Section: Quaternary Structure Of Outer Membrane Protein Oprgmentioning

confidence: 97%

“…Prolines in transmembrane domains and elsewhere have been suggested to be important for substrate transport across membranes in many ␣-helical transporters (31). For instance, Pro 28 has been shown to be important for the function of lac permease (32). In case of the phenylalanine permease PheP, Pro 341 has been shown to be critical for substrate transport (33).…”

Section: Quaternary Structure Of Outer Membrane Protein Oprgmentioning

confidence: 99%

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa

Gari

Seelheim

Marsh

et al. 2018

Journal of Biological Chemistry

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Edited by Chris WhitfieldPseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded ␤-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel-loop region might be crucial for its activity. 2 The abbreviations used are: OM, outer membrane; LPS, lipopolysaccha-Figure 4. Single molecule photobleaching of OprG in lipid bilayers. A and B, bleaching steps of single fluorescent protein complexes in lipid bilayers reveal the numbers of Alexa 647-labeled subunits. Examples of single-step (A) and two-step (B) bleaching traces are shown. The black lines represent mean intensity values from 5 ϫ 5 pixel 2 regions. The red lines represent the Chung-Kennedy nonlinear filtered versions of the data. C, average fractions of observed bleaching steps from 10 movies of two different protein reconstitutions. Division of bars into blue, green, and red illustrate the calculated fractions of monomers, dimers, and trimers within one observed population, as explained under "Experimental procedures." D, total oligomeric fractions of monomers (blue), dimers (green), and trimers (red) are calculated from data in C. Quaternary structure of outer membrane protein OprG

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Preparative refolding of small monomeric outer membrane proteins

Cited by 10 publications

References 55 publications

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Selective ¹H–¹H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa

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Preparative refolding of small monomeric outer membrane proteins

Cited by 10 publications

References 55 publications

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Probing Membrane Protein Insertion into Lipid Bilayers by Solid‐State NMR

Selective 1H–1H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa

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Selective ¹H–¹H Distance Restraints in Fully Protonated Proteins by Very Fast Magic-Angle Spinning Solid-State NMR