Strategy for membrane protein crystallization exemplified with OmpA and OmpX

Pautsch, Alexander; Vogt, Joachim; Model, Kirstin; Siebold, Christian; Schulz, Georg E.

doi:10.1002/(sici)1097-0134(19990201)34:2<167::aid-prot2>3.0.co;2-h

Cited by 96 publications

(60 citation statements)

References 29 publications

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“…1a) were synthesized in our laboratory by grafting either hydrogenated or deuterated (Ն99%) octylamine and isopropylamine groups onto a hydrogenated poly(acrylic acid) precursor (22,25 (34) were similar to those described in ref. 27. Yields were Ϸ25 mg of pure tOmpA per liter of culture.…”

Section: Methodssupporting

confidence: 77%

See 1 more Smart Citation

NMR study of a membrane protein in detergent-free aqueous solution

Zoonens

Catoire

Giusti

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

107

169

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One of the major obstacles to membrane protein (MP) structural studies is the destabilizing effect of detergents. Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep MPs water-soluble under mild conditions. In the present work, we have explored the feasibility of studying the structure of APol-complexed MPs by NMR. As a test MP, we chose the 171-residue transmembrane domain of outer MP A from Escherichia coli (tOmpA), whose x-ray and NMR structures in detergent are known. 2 H, 15 N-labeled tOmpA was produced as inclusion bodies, refolded in detergent solution, trapped with APol A8-35, and the detergent removed by adsorption onto polystyrene beads. The resolution of transverse relaxation-optimized spectroscopy-heteronuclear single-quantum correlation spectra of tOmpA͞A8-35 complexes was found to be close to that of the best spectra obtained in detergent solutions. The dispersion of chemical shifts indicated that the protein had regained its native fold and retained it during the exchange of surfactants. MP-APol interactions were mapped by substituting hydrogenated for deuterated A8-35. The resulting dipolar broadening of amide proton linewidths was found to be limited to the ␤-barrel region of tOmpA, indicating that A8-35 binds specifically to the hydrophobic transmembrane surface of the protein. The potential of this approach to MP studies by solution NMR is discussed. membrane proteins ͉ amphipols ͉ OmpA ͉ surfactant I ntegral membrane proteins (MPs) are involved in such essential cell functions as energy transduction, import and export of nutrients and drugs, signal detection, cell-to-cell communication, etc. They comprise 20-30% of the proteins encoded in the genome of cells and a majority of the targets of currently marketed drugs (1). A detailed knowledge of their structure is essential to understanding their function and dysfunction, as well as to a wide range of biomedical and biotechnological applications. The scarcity of high-resolution MP structures (which represent Ͻ0.3% of currently available structures) can be traced to three main factors: low levels of natural abundance, difficult overexpression, and a poor stability in the presence of detergent. Detergents are generally used to handle MPs in aqueous solutions, because the highly hydrophobic character of their transmembrane surface renders MPs water-insoluble. By adsorbing onto this surface, detergents make it hydrophilic (2). However, the dissociating character of detergents, combined with the need to maintain an excess of them, frequently results in more or less rapid inactivation of solubilized MPs (3).Inactivation by detergents is a particularly serious problem in the field of solution-state NMR for the following reasons: (i) to keep highly concentrated MPs (in the mM range) from aggregating, high concentrations of detergents must generally be used (usually in the 200-to 600-mM range; see, for instance, ref. 4); (ii) high temperatures are usually resorted to, to improve the resolution of the spectra; and ...

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Section: Methodssupporting

confidence: 77%

“…As a model MP, we chose the transmembrane domain of outer MP A from Escherichia coli, tOmpA (Ϸ19 kDa), a protein that has been well studied by x-ray crystallography (26)(27)(28) and NMR spectroscopy (6,(29)(30)(31)(32).…”

mentioning

confidence: 97%

NMR study of a membrane protein in detergent-free aqueous solution

Zoonens

Catoire

Giusti

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

107

169

View full text Add to dashboard Cite

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“…Usually, the folded form of an OMP has a higher electrophoretic mobility than its denatured form. In the case of PagL, however, the folded protein has a lower electrophoretic mobility as has been observed also for a few other small OMPs, i.e., OmpA171t, OmpX, and NspA (9,10). The same mobility shift was observed on Western blot for wild-type PagL folded in vivo (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL fromPseudomonas aeruginosa

Rutten

Geurtsen²,

Lambert

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…This hypothesis is supported by the reports of the crystallisation and structure elucidation of OmpA (Pautsch and Schulz, 1998;Pautsch et al, 1999), OmpT (Vandeputte-Rutten et al, 2001), and OmpX . These membrane proteins form also type III membrane protein crystals and all are crystallised in the presence of large amounts of organic solvents.…”

Section: Crystal Packing and Crystal Growthmentioning

confidence: 99%

Detergent organisation in crystals of monomeric outer membrane phospholipase A

Snijder¹,

Timmins²,

Kalk³

et al. 2003

Journal of Structural Biology

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The structure of the detergent in crystals of outer membrane phospholipase A (OMPLA) has been determined using neutron diffraction contrast variation. Large crystals were soaked in stabilising solutions, each containing a different H 2 O=D 2 O contrast. From the neutron diffraction at five contrasts, the 12 A A resolution structure of the detergent micelle around the protein molecule was determined. The hydrophobic b-barrel surfaces of the protein molecules are covered by rings of detergent. These detergent belts are fused to neighbouring detergent rings forming a continuous three-dimensional network throughout the crystal. The thickness of the detergent layer around the protein varies from 7-20 A A. The enzymeÕs active site is positioned just outside the hydrophobic detergent zone and is thus in a proper location to catalyse the hydrolysis of phospholipids in a natural membrane. Although the dimerisation face of OMPLA is covered with detergent, the detergent density is weak near the exposed polar patch, suggesting that burying this patch in the enzymeÕs dimer interface may be energetically favourable. Furthermore, these results indicate a crucial role for detergent coalescence during crystal formation and contribute to the understanding of membrane protein crystallisation.

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Strategy for membrane protein crystallization exemplified with OmpA and OmpX

Cited by 96 publications

References 29 publications

NMR study of a membrane protein in detergent-free aqueous solution

NMR study of a membrane protein in detergent-free aqueous solution

Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL fromPseudomonas aeruginosa

Detergent organisation in crystals of monomeric outer membrane phospholipase A

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