Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells

Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells. Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature. About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte. These proteins were not removed from the membrane by extensive washing, even at high ionic strength. After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes. The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques. In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6. The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain. These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.     

Gene-dose-dependent expression of soluble mammalian cytochrome b5 in Escherichia coli

Summary A synthetic structural gene encoding a mammalian cytochrome b₅, carrying an optimised ribosomal binding sequence, was tandemly polymerised ranging from one (n=1) to six (n=6) gene copies. The gene, placed in p-ncyt under the control of the P_L promoter, transcribed mono- to hexahomocistronic mRNA, expressing one to six copies of cytochrome b₅. The expressed levels of cytochrome b₅ in Escherichia coli p-ncyt corresponded linearly with the gene dose when up to five copies were present; saturating build-up of the recombinant protein was reached at six gene copies. Cells bearing p-6cyt produced 75 g cytochrome b₅/ml of unit optical density at 600 nm culture, constituting 55% of the soluble bacterial protein. The recombinant protein accumulated predominantly in a haem-deficient, apoform, together with lesser amounts of the halocytochrome b₅. Whereas the overall expressed protein (apo and holo forms) was gene dose dependent, there was an inverse relationship between holocytochrome b₅ production and gene dose. Incubation of the thermally induced bacterial lysates with exogenous haem a converted all of the soluble apocytochrome b₅ into holocytochrome b₅ that was spectrally indistinguishable with its native counterpart. Culture supplementation with the likely metabolic precursors of haem synthesis, 5-aminolevulinic acid, glycine/succinate or glutamate, significantly alleviated the protoporphyrin deficiency during hyperproduction of cytochrome b₅ in E. coli.Correspondence to: M. A. Kaderbhai     

The identity of Escherichia coli bacterioferritin and cytochrome b1.

Two different procedures were used to prepare pure samples of 'cytochrome b1' (column chromatography) and 'bacterioferritin' (immunoprecipitation) from Escherichia coli K-12 strain CA265. Both were crystallized, and X-ray-crystallographic data were compared with those from the bacterioferritin of E. coli strain W3300 used as a standard. We conclude that 'cytochrome b1' and 'bacterioferritin' are identical.     

The solution structure of oxidized Escherichia coli cytochrome b562.

The solution structure of the oxidized, paramagnetic form of cytochrome b562 from Escherichia coli (106 amino acids) is here reported as obtained from 1653 meaningful NOEs (from a total of 2051 unique NOEs), 33 (3)JHNHalpha values, and 339 pseudocontact shifts. The structure displays the typical four-helix bundle motif, and a disordered loop between helices alpha2 and alpha3, as found in the solid state. The solution structure has a conformation intermediate between the two independent solid-state molecules, although different orientations are observed for a few residues. The magnetic susceptibility tensor is similar to that of cytochrome c, which has the same ligands, although the anisotropy is somewhat smaller. This difference in the electronic structure is consistent with the thermal accessibility in cytochrome b562 of states with S > 1/2. The structure is also compared with the solution structure of the apoprotein, and some information on the role of the cofactor on the protein folding and mobility is obtained. Helix alpha4 seems to be the most sensitive to the chemical environment in terms of structure and mobility. The pKa values affecting the hyperfine-shifted signals are also discussed. Quite intriguing is the comparison of the structure of cytochrome b562 with the available structures of cytochromes c' which display a similar folding motif and similar pKa values but very little sequence similarity.     

Beta-galactosidase fused to the hydrophobic domain of cytochrome b5 spontaneously associates with liposomes

Since liver microsomal cytochrome b5 spontaneously associates with liposomes and membranes by means of its C-terminal hydrophobic domain (HP), chimeric proteins containing HP prepared by genetic fusion might also spontaneously associate with liposomes or cellular membranes. Synthetic DNA corresponding to the hydrophobic domain of cytochrome b5 was enzymatically fused in-frame to cloned DNA corresponding to the C-terminus of the Escherichia coli enzyme, beta-galactosidase. This protein, LacZ:HP, synthesized in E. coli and purified from a crude E. coli membrane extract, was shown to spontaneously associated with liposomes, as does cytochrome b5. Association is rapid and stable in the presence of salt and high pH and the fusion protein behaves as an integral membrane protein. LacZ:HP can be readily and extensively purified from crude extracts by association with liposomes and this procedure may provide a convenient purification scheme for proteins not otherwise readily purified, for example polypeptides from cloned gene fragments to be used for antibody production. These hybrid proteins may represent a new potentially useful class of polypeptides capable of hydrophobic interactions with membranes.     

The DnaK chaperone is necessary for alpha-complementation of beta-galactosidase in Escherichia coli

We show here the involvement of the molecular chaperone DnaK from Escherichia coli in the in vivo alpha-complementation of the beta-galactosidase. In the dnaK756(Ts) mutant, alpha-complementation occurs when the organisms are grown at 30 degrees C but not at 37 or 40 degrees C, although these temperatures are permissive for bacterial growth. Plasmid-driven expression of wild-type dnaK restores the alpha-complementation in the mutant but also stimulates it in a dnaK(+) strain. In a mutant which contains a disrupted dnaK gene (DeltadnaK52::Cm(r)), alpha-complementation is also impaired, even at 30 degrees C. This observation provides an easy and original phenotype to detect subtle functional changes in a protein such as the DnaK756 chaperone, within the physiologically relevant temperature.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5-vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed.     

Crystallization of beta-galactosidase from Escherichia coli.

Two crystal forms of beta-galactosidase have been obtained from Escherichia coli. One crystal form is hexagonal space group P6222 or enantiomorph, with cell dimensions a = b = 154 A, c = 750 A. The second form is monoclinic, space group P21, with cell dimensions a = 107.9 A, b = 207.5 A, c = 509.9 A, beta = 94.7 degrees. The monoclinic form seems better suited to detailed structural analysis. The crystals are radiation-sensitive, but by using synchrotron radiation in conjunction with a long (400 mm) crystal-to-film distance it was possible to resolve the individual reflections. On the basis of crystal density measurements, there are four tetramers each of molecular weight 465,000 per asymmetric unit. The Patterson function strongly suggests that two of the tetramers are related to the other two by translation. The data are consistent with the tetramers having 222 point symmetry, but this is not proven.     

Involvement of cytochrome b5 in the cytotoxic response to Escherichia coli Lipopolysaccharide

Cytotoxic lesions, induced by Gram-negative lipopolysaccharides (LPS), occur mainly in liver where the microsomal compartment of hepatocytes is involved in the detoxification mechanisms as well as in the biosynthesis of different active metabolites.The alterations induced by LPS from E. coli 0111: 134 on cytochrome b₅ and its correlation with cytochrome P450, have been studied using an in vivo reversible endotoxic shock model and 24 h non-replicative hepatocyte monolayers.Results show that cytochrome b₅ is directly affected by LPS that induces also a membrane damage with an active release of lactate dehydrogenase (LDH). The increase of cytochrome b₅ levels may enhance the efficiency of the electron transport, thus facilitating the cytochrome P450-associate oxidations and reactions involved in the repair mechanisms of membranes.     

Electron-paramagnetic-resonance spectroscopy of Bacillus subtilis cytochrome b558 in Escherichia coli membranes and in succinate dehydrogenase complex from Bacillus subtilis membranes.

Cytochrome b558 of the Bacillus subtilis succinate dehydrogenase complex was studied by electron-paramagnetic-resonance (EPR) spectroscopy. The cytochrome amplified in Escherichia coli membranes by expression of the cloned cytochrome gene and in the succinate dehydrogenase complex immunoprecipitated from solubilized B. subtilis membranes, respectively, is shown to be low spin with a highly anisotropic (gmax approximately equal to 3.5) EPR signal. The amino acid residues most likely forming fifth and sixth axial ligands to heme in cytochrome b558 are discussed on the basis of the EPR signal and the recently determined gene sequence (K. Magnusson, M. Philips, J.R. Guest, and L. Rutberg, J. Bacteriol. 166:1067-1071, 1986) and in comparison with other b-type cytochromes.     

Efflux of beta-galactosidase products from Escherichia coli.

Several different strains of Escherichia coli were grown on a variety of carbon sources under various growth conditions. Lactose was added (usually at mid-log phase), and the concentrations of the products of beta-galactosidase action on this sugar (galactose, glucose, and allolactose) were determined at various times thereafter in the total culture and in the medium. It was found that with each strain, with all carbon sources, and under all of the conditions studied, a very large proportion of the products were found in the medium. Control studies were carried out which showed that these results were not artifacts of the method of separating the cells from the medium. The results also did not arise from the secretion of beta-galactosidase into the medium, from the diffusion of substrates and products into and out of the cells due to leaks in the membrane, or from faults in the method of sugar analysis. In addition, the results showed that there were very high levels of products inside the cells under the conditions used and that the efflux of the products was rapid. The efflux might be energetically advantageous to the cell as well as being a means of storing excess products until needed.     

The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution.

The structure of cytochrome b562 from Escherichia coli has been determined at 2.5 A resolution by x-ray diffraction methods. Protein phases were computed by the single isomorphous replacement method with anomalous scattering measurements from the native and uranyl acetate-substituted crystals. The electron density was averaged about the noncrystallographic 2-fold axis relating 2 molecules in the triclinic unit cell. The protein consists of four nearly parallel alpha helices and represents a new class of cytochrome structure. The heme group is inserted between the helices near one end of the molecule with one heme face partially exposed to solvent. The two heme ligands are histidine and methionine. The 2 phenylalanines are packed internally near the heme group, and the 2 tyrosines are on the surface, also near the heme group. The folding of the protein resembles that of hemerythrin and tobacco mosaic virus protein and shows a different topology from that of cytochrome b5, cytochrome c, or the globins.     

The action of beta-galactosidase (Escherichia coli) on allolactose.

The parameters involved in the action of beta-galactosidase (EC 3.2.1.23) (Escherichia coli) on allolactose, the natural inducer of lac operon in E. coli, were studied. At low allolactose concentrations only galactose and glucose were formed, while at high allolactose concentrations transgalactolytic oligosaccharides were also produced. Detectable amounts of lactose were not formed. The V and Km values (49.6 U/mg and 0.00120 M, respectively) indicated that allolactose is as good if not a better substrate of beta-galactosidase as lactose. The pH optimum with allolactose (7.8-7.9) as well as its activation by K+ (as compared to activation by Na+) were similar to the case with lactose as substrate. The alpha-anomer of allolactose was hydrolyzed about two times as rapidly as was the beta-anomer.     

The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.     

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b5 precursor is an exocytoplasmic post-translocational event in Escherichia coli

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane. The temporal nature of cleavage of the signal sequence during pre-protein translocation has remained elusive. Using an engineered mammalian cytochrome b₅ precursor we demonstrate that the signal peptide processing in Escherichia coli is an event that can occur after almost complete exocytoplasmic translocation of the preprotein is accomplished. We discuss implications of the findings in light of the known working model of sec-dependent pre-protein translocon.