Search

The outer membrane proteins (Omps) are key factors for bacterial survival and virulence. Among the Omps that have been structurally characterized either by X-ray crystallography or by NMR in solution, the crystal structure of OmpW stands out because three of its four extracellular loops are well defined, whereas long extracellular loops in other E. coli Omps are disordered in the crystals as well as in NMR structures. OmpW thus presented an opportunity for a detailed comparison of the extracellular loops in a β-barrel membrane protein structure in crystals and in noncrystalline milieus. Here, the polypeptide backbone conformation of OmpW in 30-Fos micelles was determined. Complete backbone NMR assignments were obtained and the loops were structurally characterized. In combination with the OmpW crystal structure, NMR line shape analyses, and (15)N{(1)H}-NOE data, these results showed that intact regular secondary structures in the loops undergo slow hinge motions at the detergent-solvent interface., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

X-ray crystallography and solution NMR of detergent-reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight-stranded transmembrane β-barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, "NMR-invisible" amino acid residues in-between the extracellular loops and the β-barrel prevented complete structural characterization. Here, we show that this NMR-invisible ring around the β-barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence-specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro-coil TROSY- and CRINEPT-type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [(2)H,(15)N]-labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Optimization of aqueous solutions of the integral membrane protein (IMP) OmpW for NMR structure determination has been monitored with micro-coil NMR, which enables the acquisition of NMR spectra using only micrograms of protein and detergent. The detergent 30-Fos (2-undecylphosphocholine) was found to yield the best 2D [(15)N, (1)H]-TROSY correlation NMR spectra of [(2)H, (15)N]-labeled OmpW. For the OmpW structure determination we then optimized the 30-Fos concentration, the sample temperature and long-time stability, and the deuteration level of the protein. Some emerging guidelines for reconstitution of β-barrel integral membrane proteins in structural biology are discussed.

Aqueous solutions of the detergent Fos-10 (n-decylphosphocholine) without and with addition of the integral membrane protein (IMP) OmpX (outer membrane protein X) have been characterized using pulsed field gradient-stimulated echo (PFG-STE) NMR experiments for measurements of translational diffusion coefficients. Effective diffusion coefficients for Fos-10 micelles in the absence of OmpX were obtained by observation of NMR signals from 10-bromodecan-1-ol that had been inserted into the micelles, and in the presence of OmpX by NMR observation of the protein. It is thus shown that solutions of Fos-10-reconstituted OmpX can be quantitatively described as a mixture of Fos-10 monomers, uniform Fos-10 micelles, and uniform OmpX-containing Fos-10 micelles, with Fos-10 monomers in fast exchange between the pools of these three species. This result establishes an avenue for efficient determination of the effective translational diffusion coefficients of IMP-containing detergent micelles based on observation of the intense detergent NMR signals, which is also applicable with unlabeled IMPs. This monitoring of the species present in a given IMP solution contributes to improved guidelines for rational selection of detergent and buffer conditions in structural studies of integral membrane proteins.

In structural biology, pulsed field gradient (PFG) NMR spectroscopy for the characterization of size and hydrodynamic parameters of macromolecular solutes has the advantage over other techniques that the measurements can be recorded with identical solution conditions as used for NMR structure determination or for crystallization trials. This paper describes two transverse-relaxation-optimized (TRO) (15)N-filtered PFG stimulated-echo (STE) experiments for studies of macromolecular translational diffusion in solution, (1)H-TRO-STE and (15)N-TRO-STE, which include CRINEPT and TROSY elements. Measurements with mixed micelles of the Escherichia coli outer membrane protein X (OmpX) and the detergent Fos-10 were used for a systematic comparison of (1)H-TRO-STE and (15)N-TRO-STE with conventional (15)N-filtered STE experimental schemes. The results provide an extended platform for evaluating the NMR experiments available for diffusion measurements in structural biology projects involving molecular particles with different size ranges. An initial application of the (15)N-TRO-STE experiment with very long diffusion delays showed that the tedradecamer structure of the 800 kDa Thermus thermophilus chaperonin GroEL is preserved in aqueous solution over the temperature range 25-60 °C.

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized beta-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly (2)H, (15)N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [ (15)N, (1)H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX.