High-resolution structure of the OmpA membrane domain

Pautsch, Alexander; Schulz, Georg E.

doi:10.1006/jmbi.2000.3671

Cited by 280 publications

(250 citation statements)

References 35 publications

Supporting

Mentioning

233

Contrasting

Unclassified

Order By: Relevance

“…To further explore the dependence of D on the radius R, two ␤-barrel proteins, OmpA (20) and OprM (21), along with BR, were inserted in dodecane-free bilayers of C 12 E 5 . Compared with the diffusion of L 12 , the protein diffusion constant is significantly reduced.…”

Section: Resultsmentioning

confidence: 99%

Lateral mobility of proteins in liquid membranes revisited

Gambin

López-Esparza

Reffay

et al. 2006

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

352

377

View full text Add to dashboard Cite

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrü ck predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoylsn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D ؔ 1͞R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.bilayers ͉ transmembrane proteins ͉ diffusion ͉ peptides ͉ sponge phase I n the hydrodynamic model of Saffman and Delbrück (1), transmembrane peptides and proteins are described as diffusing in a perfectly continuous medium, ignoring the finite size of the lipids. This model predicts that the diffusion coefficient D of a simple cylinder embedded in a thin sheet of fluid matching its height ( Fig. 1) is given byIn this expression, the adjustable parameters are by order of importance: the thickness h and viscosity m of the liquid membrane, the radius R of the diffusing cylinder, and the viscosity of the surrounding aqueous phase w . This result follows from solving the flow field in the membrane and in the surrounding fluid, assuming no-slip boundary conditions at the surface of the cylinder, which is considered as large compared with the bilayer components (i.e., R Ͼ h). Numerous biological studies, both in model systems (2-4) and living cells (5, 6), refer to this continuum approach (7). Because D depends only weakly on R, the characterization of protein or rafts radii is delicate (8); for example, increasing the radius from 10 to 100 Å changes the mobility by a mere 30% [for h ϭ 30 Å and m ϭ 10 poise (P; 1 P ϭ 0.1 Pa⅐s)].To check the applicability of the Saffman-Delbrück formula (Eq. 1), we have used fringe pattern photobleaching under the microscope (9) to measure precisely the self-diffusion of transmembrane peptides and proteins of well characterized dimensions. Results and DiscussionThe weight of the bilayer thickness, h, has never been investigated. Rather than using lipids of various lengths, we opted for a unique system where the bilayer thickness can be continuously tuned, leaving the bilayer viscosity constant.We use a phase of model bilayers made of nonionic ...

show abstract

Section: Resultsmentioning

confidence: 99%

Lateral mobility of proteins in liquid membranes revisited

Gambin

López-Esparza

Reffay

et al. 2006

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

352

377

View full text Add to dashboard Cite

show abstract

“…Using this method, previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers can be detected, trapped and characterized. Three membrane-bound intermediates have been described, in which the average distances of the Trps from the bilayer center are 14-16 Å, 10-11 Å and 0-5 Å, respectively [62] [5,6,69] and according to experiments with single Trp mutants of OmpA [61] (see below), Trp-7 has to remain in the first leaflet of the lipid bilayer, while the other Trps have to be translocated across the bilayer to the second leaflet. When folding is monitored by KTSE experiments at 28-30°C, a 32-kDa band can be observed in the first few minutes of the OmpA folding reaction [30].…”

Section: Identification and Characterization Of Membranebound Foldingmentioning

confidence: 99%

“…An even number of transmembrane b strands ranging from 8 to 22 has been observed in b-barrel membrane proteins of known crystal structure. Examples are the bacterial outer membrane proteins (OMPs), such as outer membrane protein A (OmpA) with 8 b strands (b-s) [5,6], OmpT (10 b-s) [7], OmPlA (12 b-s) [8], OmpF (16 b-s), maltose (LamB) [9,10] or sucrose (ScrY) [11] porins (18 b-s), and the iron transporters FhuA [12,13] or FepA [14] (22 b-s) (see fig. 1 for some examples).…”

Section: Introductionmentioning

confidence: 99%

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

Kleinschmidt¹

2003

Cellular and Molecular Life Sciences (CMLS)

View full text Add to dashboard Cite

Abstract. The biophysical principles and mechanisms by which membrane proteins insert and fold into a biomembrane have mostly been studied with bacteriorhodopsin and outer membrane protein A (OmpA). This review describes the assembly process of the monomeric outer membrane proteins of Gram-negative bacteria, for which OmpA has served as an example. OmpA is a two-domain outer membrane protein composed of a 171-residue eight-stranded b-barrel transmembrane domain and a 154-residue periplasmic domain. OmpA is translocated in an unstructured form across the cytoplasmic membrane into the periplasm. In the periplasm, unfolded OmpA is kept in solution in complex with the molecular chaperone Skp. After binding of periplasmic lipopolysaccharide, OmpA insertion and folding occur spontaneously upon interaction of the complex with the phospholipid bilayer. Insertion and folding of the b-barrel transmembrane domain into the lipid bilayer are highly synchronized, i.e. the formation of large amounts of b-sheet secondary structure and b-barrel tertiary structure take place in parallel with the same rate constants, while OmpA inserts into the hydrophobic core of the membrane. In vitro, OmpA can successfully fold into a range of model membranes of very different phospholipid compositions, i.e. into bilayers of lipids of different headgroup structures and hydrophobic chain lengths. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers. Their formation was monitored by time-resolved distance determinations by fluorescence quenching, and they were structurally distinguished by the relative positions of the five tryptophan residues of OmpA in projection to the membrane normal. Recent studies indicate a chaperone-assisted, highly synchronized mechanism of secondary and tertiary structure formation upon membrane insertion of b-barrel membrane proteins such as OmpA that involves at least three structurally distinct folding intermediates.

show abstract

“…To date, there are six NMR solution structures of b-barrel membrane proteins [1][2][3][4][5][6] (compared to the 89 unique b-barrel structures deposited in the Protein Data Bank), all except for OprH have corresponding X-ray crystal structures. [7][8][9][10][11][12] The first b-barrel membrane protein structure determined with solution NMR was in 2001 4 and the latest structure was determined in 2011 3 yielding an average of 0.6 structures a year. The dearth of solution NMR membrane protein structures is likely due to the difficulty of obtaining quality NMR spectra, which interferes with assigning the resonances and obtaining quality structural restraints.…”

Section: Introductionmentioning

confidence: 99%

“…18,19 Other methods, such as residual dipolar couplings 20 and paramagnetic relaxation enhancement, 21 have provided additional structural restraints crucial for large a-helical proteins (e.g., a-helical membrane proteins in detergent micelles). With these advances, six solution NMR b-barrel membrane protein structures (varying in the number of b-strands (8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) and molecular weight (16-31 kDa) have been determined. [1][2][3][4][5][6] In this study, the difficulties of assigning Opa 60 , a b-barrel membrane protein from Neisseria gonorrhoeae, 22 are outlined and strategies for circumventing these challenges are demonstrated.…”

Section: Introductionmentioning

confidence: 99%

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Fox

Columbus

2013

Protein Science

View full text Add to dashboard Cite

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.

show abstract

High-resolution structure of the OmpA membrane domain

Cited by 280 publications

References 35 publications

Lateral mobility of proteins in liquid membranes revisited

Lateral mobility of proteins in liquid membranes revisited

Membrane protein folding on the example of outer membrane protein A of Escherichia coli

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Contact Info

Product

Resources

About