Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-β-β-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel β-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the β-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.     

Average thickness of the gas vesicle wall in Anabaena flos-aquae.

The average thickness of the layer of protein which forms the wall of the gas vesicles in Anabaena flos-aquae was estimated from measurements of their density and geometry. The volume of the gas space in a purified gas vesicle suspension was determined from the contraction which occurred when the gas vesicles were collapsed by pressure. The volume of the protein in the same sample was calculated from its dry weight and density. From knowledge of the geometry of the average gas vesicle the thickness of the protein layer, 1.54 nm, was then calculated. By a similar method the thickness of the Microcystis gas vesicle wall, 1.62 nm, was calculated from data published by others. The average thickness of the protein layer is, as expected, slightly less than the stacking periodicity of collapsed gas vesicle walls indicated by X-ray diffraction studies.Anabaena gas vesicles with a mean length of 494 nm have an average density of 0.119 mg μl^?1 1 mg of protein is present in gas vesicles having a, total volume of 8.43 μl and a gas space of 7.67 μl. Suspensions of isolated gas vesicles with a gas space concentration of 1 μl ml^?1 give a pressure-sensitive optical density, E_1cm (500 nm) of 2.72, but gas vacuoles in cells give a smaller value.     

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii

Gas vesicles are proteinaceous, gas‐filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in‐silico 3D‐model of GvpA of the predicted coil‐α1‐β1‐β2‐α2‐coil structure is available and implies that the two β‐chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + A_mut transformants for their ability to form gas vesicles (Vac⁺ phenotype). In most cases, an alanine substitution of a non‐polar residue did not abolish gas vesicle formation, but the replacement of single non‐polar by charged residues in β1 or β2 resulted in Vac^– transformants. A replacement of residues near the β‐turn altered the spindle‐shape to a cylindrical morphology of the gas vesicles. Vac^– transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt‐bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac^– transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid‐state NMR.     

Solid-State NMR Characterization of Gas Vesicle Structure

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.     

The accessory gas vesicle protein GvpM of haloarchaea and its interaction partners during gas vesicle formation

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M_GFP contained a single fluorescent aggregate per cell, whereas cells containing L_GFP or H_GFP were fully fluorescent. The soluble L_GFP formed stable co-aggregates with GvpM in L_GFPM transformants, but the presence of GvpH resulted in the absence of M_GFP foci in HM_GFP transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44–48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac^? transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.     

Distribution, formation and regulation of gas vesicles

A range of bacteria and archaea produce intracellular gas-filled proteinaceous structures that function as flotation devices in order to maintain a suitable depth in the aqueous environment. The wall of these gas vesicles is freely permeable to gas molecules and is composed of a small hydrophobic protein, GvpA, which forms a single-layer wall. In addition, several minor structural, accessory or regulatory proteins are required for gas vesicle formation. In different organisms, 8-14 genes encoding gas vesicle proteins have been identified, and their expression has been shown to be regulated by environmental factors. In this Review, I describe the basic properties of gas vesicles, the genes that encode them and how their production is regulated. I also discuss the function of these vesicles and the initial attempts to exploit them for biotechnological purposes.     

包封膜蛋白的仿生细胞膜

本文概述了脂质囊泡的组成成分和制作方法以及用于膜蛋白方面研究的相关技术,包括膜蛋白整合到囊泡的方法、复合体系的表征等。脂质囊泡可以为膜蛋白提供类似体内的环境,包括疏水区和内外亲水环境,因其组分单一,可以方便地进行结构、功能、信号转导等方面的研究,因此可以模拟细胞膜作为研究膜蛋白的有力工具,目前大多是以脂质体形态作为仿生囊泡体系进行这方面研究。     

Structure of Staphylococcus aureus EsxA suggests a contribution to virulence by action as a transport chaperone and/or adaptor protein

Staphylococcus aureus pathogenesis depends on a specialized protein secretion system (ESX-1) that delivers a range of virulence factors to assist infectivity. We report the characterization of two such factors, EsxA and EsxB, small acidic dimeric proteins carrying a distinctive WXG motif. EsxA crystallized in triclinic and monoclinic forms and high-resolution structures were determined. The asymmetric unit of each crystal form is a dimer. The EsxA subunit forms an elongated cylindrical structure created from side-by-side α-helices linked with a hairpin bend formed by the WXG motif. Approximately 25% of the solvent accessible surface area of each subunit is involved in interactions, predominantly hydrophobic, with the partner subunit. Secondary-structure predictions suggest that EsxB displays a similar structure. The WXG motif helps to create a shallow cleft at each end of the dimer, forming a short β-sheet-like feature with an N-terminal segment of the partner subunit. Structural and sequence comparisons, exploiting biological data on related proteins found in Mycobacterium tuberculosis, suggest that this family of proteins may contribute to pathogenesis by transporting protein cargo through the ESX-1 system exploiting a C-terminal secretion signal and/or are capable of acting as adaptor proteins to facilitate interactions with host receptor proteins.     

Insights into the Mechanisms of Membrane Curvature and Vesicle Scission by the Small GTPase Sar1 in the Early Secretory Pathway

The small GTPase protein Sar1 is known to be involved in both the initiation of COPII-coated vesicle formation and scission of the nascent vesicle from the endoplasmic reticulum. The molecular details for the mechanism of membrane remodeling by Sar1 remain unresolved. Here, we show that Sar1 transforms synthetic liposomes into structures of different morphologies including tubules and detached vesicles. We demonstrate that Sar1 alone is competent for vesicle scission in a manner that depends on the concentration of Sar1 molecules occupying the membrane. Sar1 molecules align on low-curvature membranes to form an extended lattice. The continuity of this lattice breaks down as the curvature locally increases. The smallest repeating unit constituting the ordered lattice is a Sar1 dimer. The three-dimensional structure of the Sar1 lattice was reconstructed by substituting spherical liposomes with galactoceramide lipid tubules of homogeneous diameter. These data suggest that Sar1 dimerization is responsible for the formation of constrictive membrane curvature. We propose a model whereby Sar1 dimers assemble into ordered arrays to promote membrane constriction and COPII-directed vesicle scission.     

Gas vesicles are strengthened by the outer-surface protein,GvpC

The critical collapse pressure of gas vesicles isolated from Anabaena flos-aquae decreased from 0.557 to 0.190 MPa when GvpC, the hydrophilic 22 kDa protein present on the outer surface of the gas vesicle, was removed by rising in 6 M urea. Recombinant GvpC was purified from inclusion bodies, produced in an E. coli strain containing an expression vector bearing the gene ecoding GvpC from A. flos-aquae, and then solubilised in 6 M urea. This recombinant GvpC became bound to gas vesicles that had been stripped of their native protein, when the urea was removed by dialysis; the amount which bound increased with the concentration of GvpC present. The critical pressure of these reconstituted gas vesicles increased to 0.533 MPa, 96% of the original value. These results indicate that the function of GvpC is to increase the strength of the structure.Non-standard abbreviations SBTI Soy bean trypsin inhibitor - Gvp Gas vesicle protein - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis     

The protein encoded by gvpC is a minor component of gas vesicles isolated from the cyanobacteria Anabaena flos-aquae and Microcyctis sp.

The proteins present in gas vesicles of the cyanobacteria Anabaena flos-aquae and Microcystis sp. were separated by SDS-polyacrylamide gel electrophoresis. Each contained a protein of Mr 22K whose N-terminal amino acid sequences showed homology with that of the Calothrix sp. PCC 7601 gvpC gene product. The gvpC gene from A. flos-aquae was cloned and sequenced. The derived amino acid sequence for the gene product indicated a protein, GVPc, of 193 residues and Mr 21985 containing five highly conserved 33 amino acid repeats. The sequence was identical at the N-terminus to that of the Mr 22K protein present in gas vesicles and showed correspondence to seven tryptic peptides isolated from gas vesicles. This establishes that GVPc forms a second protein component of the gas vesicle, in addition to the main constituent, the 70 residue GVPa. Quantitative amino acid analysis of entire gas vesicles reveals that GVPc accounts for only 2.9% of the protein molecules and 8.2% of the mass present: this is insufficient to form the conical end caps of the gas vesicles. It is suggested that GVPc provides the hydrophilic outer surface of the gas vesicle wall; the 33 amino acid repeats may interact with the periodic structure provided by GVPa.     

Antibodies to the N-terminal sequence of GVPa bind to the ends of gas vesicles.

Antibodies were raised against intact gas vesicles of Anabaena flos-aquae, and against a synthetic peptide (GVPaNT) whose sequence is identical to the N-terminal region of the main gas vesicle protein, GVPa. A two-stage centrifugation procedure is described for separating gold-labelled antibodies bound to gas vesicles from unbound antibodies. The GVPaNT antibody bound to gas vesicles that had been previously rinsed with SDS to remove the outer gas vesicle protein, GVPc. Treatment with this antibody caused the gas vesicles to aggregate together end-to-end rather than side-by-side. The binding of the anti-GVPaNT-immunogold particles to the gas vesicle was restricted to the conical ends of the structure. These observations indicate that the sequence to which the GVPaNT antibodies were raised, residues 1 to 13 of the GVPa molecule, is exposed only at the outer surface of the cones and that it is normally obscured by GVPc. As GVPa forms both the conical ends and the cylindrical midsection of the gas vesicle, exposure of the N-terminal sequence only in the cones must be due to differences in the contact between adjacent GVPa molecules in the central cylinders and end-cones.     

Properties of deoxycholate solubilized sarcoplasmic reticulum Ca2+-ATPase.

The Ca2+-dependent ATPase of sarcoplasmic reticulum after solubilization with deoxycholate and removal of lipid by gel chromatography exists as a mixture of monomer, dimer, and smaller amounts of higher molecular weight aggregates. The binding capcity of deoxycholate by monomeric and oligomeric forms of the ATPase is 0.3 g/g of protein at pH 8 and ionic strength 0.11. Examination in the analytical ultracentrifuge results in estimates of protein molecular weight of monomer of 115 000 +/- 7000 and of Stokes radius of 50-55 A. The results indicate an asymmetric shape of both delipidated monomer and dimer. Solubilization of ATPase vesicles by deoxycholate at high protein dilutions leads to almost instantaneous loss of ATPase activity. However, ATPase may be solubilized by deoxycholate in presence of phospholipid and sucrose in a temporarily active state. Inactivation appears to be accompanied by delipidation and conformational changes of the protein as evidenced by circular dichroism measurements. Sedimentation velocity examination of enzymatically active preparations of soluble ATPase in presence of phospholipid and sucrose strongly suggests that the major part of enzymatic activity is derived from a monomer with an asymmetric shape. The extent of formation of soluble oligomers by column chromatography was dependent on the exact conditions used for initial solubilization of ATPase. No evidence for differences among monomer and dimer fractions was obtained by isoelectric focusing and amino acid analysis. The results of these studies are compatible with electron-microscopic studies by other authors which suggest that the ATPase has an elongated shape with limited hydrophobic contact with the membrane lipid. A resemblance of delipidated oligomers with the form in which ATPase occurs in the membrane is conjectural at present.     

Interactions of the GM2 Activator Protein with Phosphatidylcholine Bilayers: A Site-Directed Spin-Labeling Power Saturation Study

The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.     

Calcium-induced potassium pathway in sided erythrocyte membrane vesicles.

We have characterized the asymmetric effect of Ca2+ on passive K+ permeability in erythrocyte membranes, using inside out and right-side out vesicles. Ca2+, but not Mg2+, can induce an increase in K+ uptake in inside out vesicles. The half-maximal concentration of Ca2+ required to induce the K+ uptake is 0.2 mM, and the permeability increase is not specific for K+. Thus, the Ca2+- induced permeation process in inside out vesicles is changed from that in the energy-depleted intact cell which requires only micromolar concentrations of Ca2+ and is specific for K+. Removal of spectrin had no effect on the vesicle permeability increase due to Ca2+. Studies with N-ethylmaleimide show that the vesicle channel openings is mediated by a protein and passage is controlled by sulfhydryl groups; furthermore, the Ca2+-induced vesicle pathway is distinct from the normal channel for passive K+ leak in the absence of Ca2+. The protein is sensitive to its phospholipid environment since removal of easily accessible phospholipid head groups on the cytoplasmic face of the vesicles inhibits the Ca2+ -stimulated channel opening.     

Buoyancy regulation of Microcystis flos-aquae during phosphorus-limited and nitrogen-limited growth

The dominance of gas-vacuolate cyanobacteria is often attributedto their buoyancy and to their ability to regulate buoyancyin response to environmental conditions. Changes in absolutegas vesicles volume, carbohydrate content, protein content andcolony buoyancy of Microcystis flos-aquae were investigatedduring nitrogen-limited, phosphorus-limited and nutrient-repletegrowth. When nutrient-replete, M. flos-aquae cells consistentlyhad excess gas vesicles, which provided sufficient buoyancythat the influence of daily carbohydrate changes on cells uponfloatation was negligible. However, during nitrogen-limitedgrowth, gas vesicle volume per cell decreased significantlywith nitrogen exhaustion. The maximum decrease of gas vesiclevolume was up to 84–88%. At the same time, cellular carbohydratecontent had an accumulation trend. The decrease of gas vesiclebuoyancy together with the daily increase in carbohydrate aresuggested to explain the daily changes in the cell floatation.During phosphorus-limited growth, gas vesicle volume per celldecreased slightly (maximum to 22–32%), and they stillprovided sufficient buoyancy that most cells kept floating eventhough there were significant daily carbohydrate changes. Sincenitrogen limitation caused more significant buoyancy loss thanphosphorus limitation did, surface water blooms may disappearor appear frequently in nitrogen limited water bodies whilethey may persist a longer time in phosphorus limited water bodies.The quantitative analysis in buoyancy change by gas vesicles,carbohydrate and protein suggested that long-term buoyancy regulationwas mainly determined by changes of gas vesicle volume whereasshort-term buoyancy regulation was mainly determined by carbohydrateaccumulation and consumption. Both long-term and short-termbuoyancy regulation were influenced by cell nutrient status.Furthermore, gas vesicle volume per cell and protein contentchanged in the same way in both nitrogen-limited and phosphorus-limitedgrowth, which implied that the decrease of gas vesicles wereassociated with controls of total protein synthesis.     

A convenient large-scale method for the isolation of membrane vesicles permeable to a specific inorganic ion: Isolation and characterization of functional acetylcholine receptor-containing vesicles from the electric organ of Electrophorus electricus

A convenient, large-scale method for the isolation of membrane vesicles permeable to specific inorganic ions has been developed. The general principle of this method involves the exchange of Na⁺ within the vesicles for external Cs⁺. Vesicles in which this exchange rapidly occurs can be separated on the basis of their density from vesicles in which the exchange occurs slowly (G. P. Hess and J. P. Andrews (1977) Proc. Nat. Acad. Sci. USA74, 482–486). This approach has been adapted to develop a method suitable for the large-scale isolation of vesicles that contain functional acetylcholine receptors from the Electrophorus electricus electroplax. The new procedure involves a discontinuous sucrose gradient for an initial purification of the vescles. This allows the use of a low-speed centrifuge, which has a capacity up to 30 times greater than the Beckman ultracentrifuge previously used. A self-forming CsCl-Percoll gradient and low-speed centrifugation are then used for the isolation of the functional acetylcholine receptor-containing vesicles. The isolation step leads close to the theoretically possible fourfold purification of the vesicles that contain functional receptors. The yield, up to 12 mg membrane protein/centrifugal run, is about 100-fold higher than the yield from the sucrose-CsCl density gradient previously (Hess and Andrews, see above) used. The gradients are self-forming and an equilibrium is reached after centrifugation for only 30 min. In 12 experiments with membrane preparations from 12 different ceis, the functional vesicles had an internal volume of 2.0 ± 0.3 μl/mg vesicle protein and a receptor concentration of 1.2 ± 0.02 μm (1.2 μmol/liter of internal volume). Electron micrographs of these vesicles show an average vesicle radius of 1600 ± 300 Å. From these results, an average of 12 receptor molecules/membrane vesicle is calculated.     

Isolation and characterization of gas vesicles from Microcyclus aquaticus

Intact gas vesicles of Microcyclus aquaticus S1 were isolated by using centrifugally accelerated flotation of vesicles and molecular sieve chromatography. Isolated gas vesicles were cylindrical organelles with biconical ends and measured 250×100 nm. The gas vesicle membrane was composed almost entirely of protein; neither lipid nor carbohydrate was detected, although one mole of phosphate per mole of protein was found. Amino acid analysis indicated that the protein contained 54.6% hydrophobic amino acid residues, lacked sulfur-containing amino acids, and had a low aromatic amino acid content. The protein subunit composition of the vesicles was determined by gel electrophoresis in (i) 0.1% sodium dodecyl sulfate at pH 9.0 and (ii) 5 M urea at pH 2.0. The membrane appeared to consist of one protein subunit of MW 50 000 daltons. Charge isomers of this subunit were not detected on urea gels. Antiserum prepared against purified gas vesicles of M. aquaticus S1 cross-reacted with the gas vesicles of all other gas vacuolate strains of M. aquaticus, as well as those of Prosthecomicrobium pneumaticum, Nostoc muscorum, and Anabaena flos-aquae, indicating that the gas vesicles of these widely divergent organisms have some antigenic determinants in common.Abbreviations SDS sodium dodecyl sulfate - MW molecular weight - Tris tris(hydroxymethyl)aminomethane - EDTA disodium ethylenediaminetetraacetic acid - BSA bovine serum albumin - TCA trichloroacetic acid - P _c pressure necessary to collapse gas vesicles     

The sequence of the major gas vesicle protein,GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.