Article

Micro-coil NMR to monitor optimization of the reconstitution conditions for the integral membrane protein OmpW in detergent micelles

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Journal of Biomolecular NMR (Impact Factor: 3.14). 08/2012; 54(2):129-33. DOI: 10.1007/s10858-012-9658-x
Source: PubMed

ABSTRACT

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.

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    ABSTRACT: This chapter will give a state of the art review on the most commonly used biophysical characterization techniques used in β-barrel outer membrane protein (OMP) structural analysis such as X-ray diffraction analysis of protein crystals, Circular Dichroism (CD) spectroscopy and Nuclear Magnetic Resonance (NMR). For each of the aforementioned techniques the alliances with complementary as well as with other sophisticated techniques to obtain structural, kinetic and thermodynamical information will be reported. Though X-Ray diffraction measurements and NMR studies result in valuable tertiary structure information, while CD spectroscopy can give information on the secondary structure mainly, CD will be particularly analyzed as a reliable and user friendly but under-represented method with which to obtain rapid secondary structural information on wild-type and engineered β-barrel OMPs. Protein structural information studies are especially important for genetically engineered β-barrel OMP channel variants with novel structural/geometrical features. Being as nano-material components the correct protein folding into the desired structure is a prerequisite for channel function and reconstitution ability, therefore a particular focus should be given to structure determination.
    No preview · Article · Jan 2014 · Advances in Experimental Medicine and Biology
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    ABSTRACT: 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.
    No preview · Article · May 2014 · ChemBioChem
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    ABSTRACT: 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 15N{1H}-NOE data, these results showed that intact regular secondary structures in the loops undergo slow hinge motions at the detergent-solvent interface.
    Preview · Article · Aug 2014 · Structure
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