Bio-Beads: An Efficient Strategy for Two-Dimensional Crystallization of Membrane Proteins

Rigaud, Jean‐Louis; Mosser, Gervaise; Lacapère, J; Olofsson, Anders; Lévy, Daniel; Ranck, Jean-Luc

doi:10.1006/jsbi.1997.3848

Cited by 192 publications

(135 citation statements)

References 29 publications

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“…Complex I (2 mg/ml) and the lipid solution were mixed in a 1:4 (w/w) ratio. BioBeads with a capacity of absorbing 105 mg of dodecyl maltoside per g of beads (28) were added at an 8-fold excess. The mixture was stirred for 3 h at 4°C and centrifuged for 30 min at 150,000 ϫ g (Beckmann Airfuge) after separation of the beads.…”

Section: Methodsmentioning

confidence: 99%

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

Stolpe

Friedrich

2004

Journal of Biological Chemistry

104

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The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na ؉ /H ؉ exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na ؉ /H ؉ antiport is discussed.

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Section: Methodsmentioning

confidence: 99%

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

Stolpe

Friedrich

2004

Journal of Biological Chemistry

104

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“…50 l of PilQ (100 g/ml in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, and 0.1% (w/v) Zwittergent 3-10) were mixed with 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine to provide a final protein/lipid ratio of 10:1. Hydrophobic polystyrene beads, or Bio-Beads, have been shown to provide a surface for nonspecific adsorption of detergent and are a convenient method for detergent extraction (14). Hydrated SM2 Bio-Beads (Bio-Rad) were added to the PilQ/1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine solution, and the mixture was incubated at 20°C for 24 -48 h. Large two-dimensional crystal sheets of PilQ were formed at the air-water interface of the solution; the crystals were examined using TEM by gently applying glow-discharged carbon-coated copper grids to the surface of the solution, followed by negative staining with 4% (w/v) uranyl acetate as described previously (11).…”

Section: Two-dimensional Crystallization Of Pilq Andmentioning

confidence: 99%

Structure of the Neisseria meningitidis Outer Membrane PilQ Secretin Complex at 12 Å Resolution

Collins

Frye

Kitmitto

et al. 2004

Journal of Biological Chemistry

118

123

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The bacterial pathogen Neisseria meningitidis expresses long, thin, retractile fibers (called type IV pili) from its cell surface and uses these adhesive structures to mediate primary attachment to epithelial cells during host colonization and invasion. PilQ is an outer membrane protein complex that is essential for the translocation of these pili across the outer membrane. Here, we present the structure of the PilQ complex determined by cryoelectron microscopy to 12 Å resolution. The dominant feature of the structure is a large central cavity, formed by four arm features that spiral upwards from a squared ring base and meet to form a prominent cap region. The cavity, running through the center of the complex, is continuous and is effectively sealed at both the top and bottom. Analysis of the complex using selforientation and by examination of two-dimensional crystals indicates a strong C4 rotational symmetry, with a much weaker C12 rotational symmetry, consistent with PilQ possessing true C4 symmetry with C12 quasisymmetry. We therefore suggest that the complex is a homododecamer, formed by association of 12 PilQ polypeptide chains into a tetramer of trimers. The structure of the PilQ complex, with its large and well defined central chamber, suggests that it may not function solely as a passive portal in the outer membrane, but could be actively involved in mediating pilus assembly or modification.

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“…The method of detergent removal by hydrophobic adsorption onto polystyrene beads, previously shown to be very efficient for proteoliposome reconstitution (24), has been recently demonstrated to be a powerful alternative to conventional dialysis for 2-D crystallization trials (40). Using radioactive detergents, the method has been calibrated precisely in terms of the adsorptive capacity of the beads and the rates of detergent removal.…”

Section: -D Crystalsmentioning

confidence: 99%

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

2002

Braz J Med Biol Res

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Reconstitution of membrane proteins into lipid bilayers is a powerful tool to analyze functional as well as structural areas of membrane protein research. First, the proper incorporation of a purified membrane protein into closed lipid vesicles, to produce proteoliposomes, allows the investigation of transport and/or catalytic properties of any membrane protein without interference by other membrane components. Second, the incorporation of a large amount of membrane proteins into lipid bilayers to grow crystals confined to two dimensions has recently opened a new way to solve their structure at high resolution using electron crystallography. However, reconstitution of membrane proteins into functional proteoliposomes or 2-D crystallization has been an empirical domain, which has been viewed for a long time more like "black magic" than science. Nevertheless, in the last ten years, important progress has been made in acquiring knowledge of lipid-protein-detergent interactions and has permitted to build upon a set of basic principles that has limited the empirical approach of reconstitution experiments. Reconstitution strategies have been improved and new strategies have been developed, facilitating the success rate of proteoliposome formation and 2-D crystallization. This review deals with the various strategies available to obtain proteoliposomes and 2-D crystals from detergent-solubilized proteins. It gives an overview of the methods that have been applied, which may be of help for reconstituting more proteins into lipid bilayers in a form suitable for functional studies at the molecular level and for high-resolution structural analysis. Correspondence

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Bio-Beads: An Efficient Strategy for Two-Dimensional Crystallization of Membrane Proteins

Cited by 192 publications

References 29 publications

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

The Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I) Is a Primary Proton Pump but May Be Capable of Secondary Sodium Antiport

Structure of the Neisseria meningitidis Outer Membrane PilQ Secretin Complex at 12 Å Resolution

Membrane proteins: functional and structural studies using reconstituted proteoliposomes and 2-D crystals

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