Pseudomonas glumae lipase: increased proteolytic stability by protein engineering.

The feasibility of stabilizing proteins towards proteolytic degradation was explored by engineering the primary proteolytic cleavage site(s). This novel approach does not require information on the 3-D structure of the native enzyme. As a model system, the extracellular lipase of Pseudomonas glumae was chosen, which is sensitive towards degradation by subtilisin-type proteases. The primary proteolytic cleavage in the lipase appeared to be located between amino acids serine 153 and histidine 154. Since subtilisins are known to show a preference towards amino acid residues surrounding the scissile bond, non-preferred amino acids were introduced in this area. Two concepts were tested: the introduction of arginine or glutamate residues (charge concept) and the introduction of proline residues (proline concept). Although the mutant lipases produced according to either of these concepts were still cleaved in the same area, they showed a considerably increased stability towards proteolytic degradation.

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.

Lipid involvement in protein translocation in Escherichia coli.

Signal peptides play an essential role in protein translocation. This review summarizes the current knowledge of the structure of signal peptides and signal peptide-lipid interactions and addresses the possibility that signal peptide-lipid interactions initiate membrane translocation of precursor proteins. A new model for protein translocation in Escherichia coli is proposed, which includes as central features conformational changes of the signal peptide and signal-peptide-induced local changes in membrane organization (non-bilayer lipids).

Inhibition of PhoE translocation across Escherichia coli inner-membrane vesicles by synthetic signal peptides suggests an important role of acidic phospholipids in protein translocation.

To obtain insight into the mechanism of precursor protein translocation across membranes, the effect of synthetic signal peptides and other relevant (poly)peptides on in vitro PhoE translocation was studied. The PhoE signal peptide, associated with inner membrane vesicles, caused a concentration-dependent inhibition of PhoE translocation, as a result of a specific interaction with the membrane. Using a PhoE signal peptide analog and PhoE signal peptide fragments, it was demonstrated that the hydrophobic part of the peptide caused the inhibitory effect, while the basic amino terminus is most likely important for an optimal interaction with the membrane. A quantitative analysis of our data and the known preferential interaction of synthetic signal peptides with acidic phospholipids in model membranes strongly suggest the involvement of negatively charged phospholipids in the inhibitory interaction of the synthetic PhoE signal peptide with the inner membrane. The important role of acidic phospholipids in protein translocation was further confirmed by the observation that other (poly)peptides, known to have both a high affinity for acidic lipids and hydrophobic interactions with model membranes, also caused strong inhibition of PhoE translocation. The implication of these results with respect to the role of signal peptides in protein translocation is indicated.

Modulation of membrane surface curvature by peptide-lipid interactions.

Recent reports on the interaction of cardiotoxin and melittin with phospholipid model membranes are reviewed and analyzed. These types of peptide toxins are able to modulate lipid surface curvature and polymorphism in a highly lipid-specific way. It is demonstrated that the remarkable variety of effects of melittin on the organization of different membrane phospholipids can be understood in a relatively simple model, based on the shape-structure concept of lipid polymorphism and taking into account the position of the peptide molecule with respect to the lipids. Based on the strong preference of the peptides for negatively charged lipids and the structural consequences thereof, and on preliminary studies of signal peptide-lipid interaction, a role of inverted or concave lipid structures in the process of protein translocation across membranes is suggested.

Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization.

In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.

Melittin-induced changes of the macroscopic structure of phosphatidylethanolamines.

The binding of melittin to phosphatidylethanolamine model systems and its influence on the supramolecular organization of the lipid were investigated with binding assays, differential scanning calorimetry, 31P NMR, freeze-fracture electron microscopy, and small-angle X-ray scattering. The results are compared with binding to an analogous phosphatidylcholine and structural consequences thereof. Melittin binds with similar affinity to both lipid types in the liquid-crystalline state; at gel-phase temperatures, in contrast, interaction with phosphatidylethanolamine is much weaker and does not lead to the bilayer fragmentation observed for phosphatidylcholines. With regard to phosphatidylethanolamine polymorphism, it is shown that melittin acts as an inhibitor of HII-phase formation and as a stabilizer of the bilayer organization. It is demonstrated that the remarkable variety of effects of melittin on the polymorphism of different membrane phospholipids can be understood in a relatively simple concept, taking into account the relative position and the shape of the interacting components.

Characterization of the interfacial behavior and structure of the signal sequence of Escherichia coli outer membrane pore protein PhoE.

The behavior of the chemically synthesized PhoE signal peptide and signal peptide fragments on hydrophilic-hydrophobic interfaces was studied with circular dichroism and monolayer techniques. The experimental results were compared with computer-calculated predictions of peptide structure, orientation, and molecular area. The complete signal sequence was found to aggregate in a beta-sheet structure when introduced in an aqueous environment; on the other hand, in sodium dodecyl sulfate micelles approximately 75% alpha-helical structure was observed. Assuming this to reflect the actual structure in a peptide monolayer and taking into account the orientations predicted for the fragments, the measured molecular areas suggest a looped orientation of the signal sequence with both N and C terminus in the water phase.

Interaction of melittin with negatively charged phospholipids: consequences for lipid organization.

A characterization of the structural alterations induced by melittin in model-membranes of dioleoylphosphatidic acid and egg phosphatidylglycerol is presented, based on the use of 31P-NMR, freeze-fracture electron microscopy and small angle X-ray scattering. In accordance with earlier findings on the cardiolipin-melittin system, melittin is found to have an inverted phase inducing effect on these negatively charged lipids, in contrast to the influence on zwitterionic phospholipids. In phosphatidic acid this is expressed in the formation of an HII phase; in phosphatidylglycerol a less ordered, non-lamellar structure with low water content is adopted.

Melittin induces HII phase formation in cardiolipin model membranes.

The interaction of melittin with bovine heart cardiolipin model membranes was investigated via binding assays, 31P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and fluorescence based fusion assays. A strong binding (Kd less than 10(-7) M) appeared to be accompanied by the formation of large structures, resulting from a fusion process of extremely fast initial rate. As the melittin content is increased, bilayer structure is gradually lost and from a cardiolipin to melittin ratio of about 6 the lipid starts to organize itself in an hexagonal HII phase. At lower temperatures (T less than 40 degrees C) the coexistence of another structure is observed, characterized by a broad isotropic 31P-NMR signal and giving rise to sharp X-ray reflections, most probably a cubic phase, as suggested also be freeze-fracture images, showing orderly stacked particles. The results are discussed in relation to contrasting observations on the structural changes induced by melittin in the zwitterionic phospholipid system of dipalmitoylphosphatidylcholine (Dufourcq. J. et al. (1986) Biochim. Biophys. Acta 859, 33-48). The biological relevance of the observations with respect to the process of protein insertion into membranes is indicated.

Lipid specific penetration of melittin into phospholipid model membranes.

The relative depth of penetration of melittin into egg phosphatidylcholine and bovine heart cardiolipin model membranes was investigated using fluorescence spectroscopy techniques. The tryptophan intrinsic fluorescence shift suggests a more hydrophobic surrounding of this residue in cardiolipin, while the accessibility for charged and uncharged aqueous quenchers is decreased in the cardiolipin system when compared with the phosphatidylcholine-bound situation. A lipid incorporated hydrophobic, collisional quencher and a resonance energy transfer acceptor on the other hand are more effective in quenching the tryptophan fluorescence of cardiolipin bound melittin. The combination of these results is interpreted as prove of a deeper positioning of the tryptophan containing part of the peptide molecule in the cardiolipin system in comparison with the situation in phosphatidylcholine. Models that take this difference into account are presented, which try to explain the opposite effect of melittin binding to the two lipid systems with respect to supramolecular structure, as reported in the preceding article (Batenburg, A.M., Hibbeln, J.C.L., Verkleij, A.J. and De Kruijff, B. (1987) Biochim. Biophys. Acta 903, 142-154).

Penetration of a cardiotoxin into cardiolipin model membranes and its implications on lipid organization.

The interaction of cardiotoxin II of Naja mossambica mossambica with cardiolipin model membranes was investigated by binding, fluorescence, resonance energy transfer, fluorescence quenching, 31P NMR, freeze-fracture, and small-angle X-ray experiments. An initially electrostatic binding appeared to be accompanied by a deep penetration, most likely into the acyl chain region of the phospholipids, indicating a hydrophobic contribution to the strong interaction (KD congruent to 5 X 10(-8) M). This binding results in a fusion of unilamellar vesicles as indicated by a fluorescence-based fusion assay, freeze-fracture, and X-ray diffraction. In these fused structures freeze-fracture electron microscopy reveals the appearance of particles, which is accompanied by the induction of an isotropic component in 31P NMR. The well-defined particles are interpreted as inverted micelles, and the localization of the cardiotoxin molecule in these structures is discussed.

The anion permeability of vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane.

Band 3 protein was reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 2500:1 phospholipid:protein molar ratio by means of a Triton X-100/beads method. The SO2-4 permeability of the resulting vesicles was measured using an influx assay procedure in which the vesicles were sampled and subsequently eluted over Sephadex columns at appropriate time intervals. The accuracy of the assay was greatly increased by using an internal standard in order to correct for vesicle recovery. In agreement with previous work, it could be demonstrated that incorporation of band 3 in the vesicles caused an increase in SO2-4 permeability, which could be (partially) inhibited by high concentrations of DIDS or a competitive anion such as thiocyanate. However, the magnitude of the increased SO2-4 permeability was highly variable, even when vesicles were reconstituted using band 3 isolated from one batch of ghosts. In addition, the SO2-4 influx curves showed complex kinetics. These results are related to the existence of vesicle heterogeneity with respect to protein content and vesicle size as revealed by stractan density gradient centrifugation and freeze-fracture electron microscopy. Band 3 incorporation also increased the L-glucose permeability of the vesicles which could also be inhibited by DIDS. Glycophorin, which has no known transport function, reconstituted with lipid vesicles consisting of 94:6 (molar ratio) egg phosphatidylcholine-bovine heart phosphatidylserine in a 400:1 phospholipid:protein molar ration increased the bilayer permeability towards SO2-4 as well as towards L-glucose. Surprisingly, the SO2-4 permeability in the vesicles could also be inhibited by DIDS and thiocyanate. It is concluded that the use of DIDS and a competitive anion, thiocyanate, in order to prove that band 3 is functionally reconstituted, is highly questionable. The increased SO2-4 and L-glucose permeability of band 3-lipid as well as glycophorin-lipid vesicles and the inhibitory action of DIDS are discussed in the light of the presence of defects at the lipid/protein interface and protein aggregation, which may induce the formation of pores. Since the band 3-lipid vesicles are more permeable for SO2-4 than for L-glucose, in contrast to the glycophorin-containing vesicles, it is suggested that some anion specificity of the increased bilayer permeability in the band 3-lipid vesicles is still preserved.