A cytochrome c fusion protein domain for convenient detection, quantification, and enhanced production of membrane proteins in Escherichia coli��Expression and characterization of cytochrome-tagged Complex I subunits

Overproduction of membrane proteins can be a cumbersome task, particularly if high yields are desirable. NADH:quinone oxidoreductase (Complex I) contains several very large membrane‐spanning protein subunits that hitherto have been impossible to express individually in any appreciable amounts in Escherichia coli. The polypeptides contain no prosthetic groups and are poorly antigenic, making optimization of protein production a challenging task. In this work, the C‐terminal ends of the Complex I subunits NuoH, NuoL, NuoM, and NuoN from E. coli Complex I and the bona fide antiporters MrpA and MrpD were genetically fused to the cytochrome c domain of Bacillus subtilis cytochrome c₅₅₀. Compared with other available fusion‐protein tagging systems, the cytochrome c has several advantages. The heme is covalently bound, renders the proteins visible by optical spectroscopy, and can be used to monitor, quantify, and determine the orientation of the polypeptides in a plethora of experiments. For the antiporter‐like subunits NuoL, NuoM, and NuoN and the real antiporters MrpA and MrpD, unprecedented amounts of holo‐cytochrome fusion proteins could be obtained in E. coli. The NuoHcyt polypeptide was also efficiently produced, but heme insertion was less effective in this construct. The cytochrome c₅₅₀ domain in all the fusion proteins exhibited normal spectra and redox properties, with an E_m of about +170 mV. The MrpA and MrpD antiporters remained functional after being fused to the cytochrome c‐tag. Finally, a his‐tag could be added to the cytochrome domain, without any perturbations to the cytochrome properties, allowing efficient purification of the overexpressed fusion proteins.     

Exploring metallodrug–protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound

Electrospray ionisation mass spectrometry was used to analyse the reactions of metal compounds with mixtures of selected proteins. Three representative medicinally relevant compounds, cisplatin, transplatin and the organometallic ruthenium compound RAPTA-C, were reacted with a pool of three proteins, ubiquitin, cytochrome c and superoxide dismutase, and the reaction products were analysed using high-resolution mass spectrometry. Highly informative electrospray ionisation mass spectra were acquired following careful optimisation of the experimental conditions. The formation of metal–protein adducts was clearly observed for the three proteins. In addition, valuable information was obtained on the nature of the protein-bound metallofragments, on their distribution among the three different proteins and on the binding kinetics. The platinum compounds were less reactive and considerably less selective in protein binding than RAPTA-C, which showed a high affinity towards ubiquitin and cytochrome c, but not superoxide dismutase. In addition, competition studies between cisplatin and RAPTA-C showed that the two metallodrugs have affinities for the same amino acid residues on protein binding. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Probing protein electrostatic interactions through temperature/reduction potential profiles

 The change in the equilibrium reduction potentials of the iron-sulfur proteins, Pyrococcus furiosus rubredoxin and P. furiosus ferredoxin, and heme protein, horse cytochrome c, has been calculated as a function of temperature using a numerical solution to the Poisson-Boltzman equation. Working curves for different internal dielectric constants were generated to best reproduce experimental observation. Based on a comparison of the experimental and simulated change in reduction potential with temperature, it is concluded that the dielectric constant of proteins is temperature-dependent and varies from protein to protein. For example, the temperature-dependent reduction potential of cytochrome c can only be simulated using a different temperature-dependent dielectric constant for each oxidation state, but this was not the case for rubredoxin or ferredoxin. The role of changes in ionization states of cytochrome c at alkaline pHs, where the reduction potential is known to be pH-dependent at room temperature, is also discussed in terms of electrostatic interaction energies as a function of temperature. It appears that temperature/reduction potential profiles may provide a direct method for measuring relative changes in internal protein dielectric constants. Received: 29 April 1996 / Accepted: 1 August 1996     

Conformational States of the Water-Soluble Fragment of Cytochrome b5. I. pH-Induced Denaturation

Cytochrome b ₅ is a membrane protein that comprises two fragments: one is water-soluble and heme-containing, and the other is hydrophobic and membrane-embedded. The function of electron transfer is performed by the former whose crystal structure is known; however, its conformational states when in the membrane field and interacting with other proteins are still to be studied. Previously, we proposed water–alcohol mixtures for modeling the effect of membrane surface on proteins, and used this approach to study the conformational behavior of positively charged cytochrome c as well as relatively neutral retinol-binding protein also functioning in the field of a negatively charged membrane. The current study describes the conformational behavior of the negatively charged water-soluble fragment of cytochrome b ₅ as dependent on pH. Decreasing pH was shown to transform the fragment state from native to intermediate, similar to the molten globule reported earlier for other proteins in aqueous solutions: at pH 3.0, the fragment preserved a pronounced secondary structure and compactness but lost its rigid tertiary structure. A possible role of this intermediate state in cytochrome b ₅ functioning is discussed.     

A Study of Protein Electrochemistry on a Supported Membrane Electrode

Protein electrochemistry offers a direct method to identify and characterize biological electron transfer processes, potentially leading to commercial applications such as biosensors and diagnostic tools. However, establishing a biocompatible electrode interface that maintains the native state of the redox protein involves several challenges. In general, membrane proteins require the presence of a phospholipid bilayer to maintain their biological activity. Synthetic `biomimetic’ membranes are widely used to characterize membrane proteins, however they have seldom been applied to measurements of protein redox activity in electrochemical cells due to their inherent insulating property. In this study we demonstrate the use of the phospholipids: PC, PC/PG and PC/PG/cholesterol membrane mixtures on chemically modified (supported) gold electrode surfaces for direct protein electrochemistry. We compare the electrochemical activity of a relatively small, redox active “test protein”, cytochrome c, in the presence and absence of phospholipid on a gold electrode modified with thiol self assembled monolayers, to explore the effect of chain length and composition of the thiol on the charge coupling. Three thiols were investigated as self assembled monolayers on a gold electrode: octanethiol, mercaptopropionic and mercaptoundecanoic acid. We demonstrate here that the charge transfer efficiency of cytochrome c is better in the presence of the membrane and in addition, a superior redox response is obtained with surfaces modified with a thiol functionalised with a carboxylic acid.On leave from: Research Group on Laser Physics of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary.Australian Peptide Conference Issue.     

Studies on the biogenesis of an enzymatically active complex III of the respiratory chain from yeast mitochondria

Complex III isolated from yeast mitochondria catalyzed an antimycin A and Diuron-sensitive coenzyme QH₂-cytochrome c reductase activity with a turnover number of 15.7 sec^?1 and contained 10 nmoles of cytochrome b and 4.6 nmoles of cytochrome c₁ per mg of protein. Electrophoresis in sodium dodecyl sulfate acrylamide gels resolved Complex III into 10 bands with apparent molecular weights of 50,000, 40,000, 30,000, 29,000, 24,000, 17,000, 16,000, 12,000, 8,400, and 5,800. Yeast cells were labeled under nongrowing conditions with (³⁵S)-methionine in the absence or presence of inhibitors of cytoplasmi? or mitochondrial protein synthesis. Labeled Complex III was isolated by immunoprecipitation from detergent-solubilized mitochondria using antiserum raised against the purified complex. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis revealed that a 30,000-dalton protein, cytochrome b, as well as 16,000-dalton protein were labeled in the presence of cycloheximide, indicating that they are products of mitochondrial protein synthesis. Immunoprecipitates from mitochondria obtained from cells labeled in the presence of chloramphenicol contained a new radioactive peak with a molecular weight of 100,000. In addition, significant decreases in the labeling of the proteins with molecular weights of 50,000, 40,000, 30,000, and 16,000 were observed. When Complex III was isolated by immunoprecipitation from intact spheroplasts after a 5-minute pulse with (³⁵S)-methionine, the 100,000-dalton protein was labeled in the immunoprecipitate whether or not chloramphenicol was present; however, after a 1-hour chase with unlabeled methionine, decreased labeling of the 100,000-dalton protein was observed concomitant with an increased labeling of the 50,000- and 40,000-dalton proteins. These results suggest that a protein with a molecular weight of 100,000 may either be a precursor or a partially assembled form of other proteins of Complex III, most probably the two largest polypeptides.     

Expression and characterization of recombinant human cytochrome c in E. coli

Cytochrome c is a heme protein involved in electron transfer, cell apoptosis, and diseases associated with oxidative stress. Here we expressed human cytochrome c in E. coli and purified it to homogeneity with a yield of 10–15 mg/L. The redox potential of recombinant human cytochrome c was 0.246 V which was measured by cyclic voltammetry. This is similar to that of horse cytochrome c with a value of 0.249 V. The sequential assignment and structural analysis of recombinant human ferrocytochrome c were obtained using multidimensional NMR spectroscopy. On the basis of our NMR studies, the recombinant human cytochrome c produced in E. coli exhibits the same tertiary fold as horse cytochrome c. These results provide evidence that human cytochrome c expressed in E. coli possesses a similar function and structure to that of the horse protein. It is known that cytochrome c plays a role in many human diseases. This study serves as the basis for gaining insight into human diseases by exploring structure and function relationships of cytochrome c to its interacting proteins.     

Proteomic maps of subcellular protein fractions of the Asian citrus psyllid Diaphorina citri,the vector of citrus huanglongbing

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is an insect vector that transmits the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) associated with the destructive citrus disease, citrus huanglongbing (HLB). Currently, D. citri is the major target in HLB management, although insecticidal control and disruption of the D. citri–CLas interactions both face numerous challenges. The present study reports the subcellular proteomic profiles of D. citri, encompassing the three main subcellular protein fractions: cytosol, mitochondria and microsomes. After optimization, subcellular proteins of both high and low abundance are obtained by two‐dimensional gel electrophoresis (2‐DE). A total of 1170 spots are detected in the 2‐DE gels of the three subcellular fractions. One hundred and sixty‐four differentially expressed proteins are successfully identified using liquid chromatography‐dual mass spectroscopy. An efficient protocol for subcellular protein fractionation from D. citri is established and a clear protein separation is achieved with the chosen protein fractionation protocol. The identified cytosolic proteins are mainly metabolic enzymes, whereas a large portion of the identified proteins in the mitochondrial and microsomal fractions are involved in ATP biosynthesis and protein metabolism, respectively. Protein–protein interaction networks are predicted for some identified proteins known to be implicated in pathogen–vector interactions, such as actin, tubulin and ATP synthase, as well as insecticide resistance, such as the cytochrome P450 superfamily. The findings should provide useful information to help identify the mechanism responsible for the CLas–D. citri interactions and eventually contribute to D. citri control.     

A Protein Structure Initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles

A specialized vector backbone from the Protein Structure Initiative was used to express full-length human cytochrome b5 as a C-terminal fusion to His8-maltose binding protein in Escherichia coli. The fusion protein could be completely cleaved by tobacco etch virus protease, and a yield of 18 mg of purified full-length human cytochrome b5 per liter of culture medium was obtained (2.3 mg per g of wet weight bacterial cells). In situ proteolysis of the fusion protein in the presence of chemically defined synthetic liposomes allowed facile spontaneous delivery of the functional peripheral membrane protein into a defined membrane environment without prior exposure to detergents or other lipids. The utility of this approach as a delivery method for production and incorporation of monotopic (peripheral) membrane proteins is discussed.     

Reconstitution of the ubiquinol: cytochrome c reductase from a bc1 subcomplex and the 'Rieske' iron-sulfur protein isolated by a new method

1. A method for preparing the 'Rieske' iron-sulfur protein and the bc1 subcomplex of complex III was developed. The new method is advantageous over the published ones in that: (a) the final yield and amount exceeds by far those obtained when employing the hitherto published methods; (b) the iron-sulfur protein as well as the bc1 subcomplex are obtained by one and the same preparation procedure from a common source; and (c) the preparation method is easier than the published ones. 2. The iron-sulfur protein obtained represents the first reconstitutively active preparation present in a monodisperse state. 3. The reconstitution of the ubiquinol:cytochrome c reductase from the two components is a reversible dissociation process. Full activity of ubiquinol:cytochrome c reductase is reached after saturation of the binding site of the bc1 subcomplex for iron-sulfur protein. 4. Full reduction of the constituent cytochrome c1 of the bc1 subcomplex can already be obtained with substoichiometric amounts of iron-sulfur protein, however. 5. The question might be raised whether the observed dissociation equilibrium represents merely a phenomenon occurring specifically with the proteins isolated in Triton X-100 and investigated in a Triton-containing buffer, or whether dissociation of the iron-sulfur protein also takes place in the mitochondrial membrane in the course of the electron-transfer reaction sequence.     

The colicin A pore-forming domain fused to mitochondrial intermembrane space sorting signals can be functionally inserted into the Escherichia coli plasma membrane by a mechanism that bypasses the Tol proteins

Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c₁ (pc1) as well as the first 167 amino acids of cytochrome b₂ (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfColA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome C₁ presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c, presequence moiety of pc1-pfColA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfColA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (ii) this channel activity was inhibited within the membrane by the immunity protein.     

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b ₅₅₄ (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b ₅₅₆ (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth. Received: 21 August 1996 / Accepted: 24 October 1996     

The singular properties of photosynthetic cytochrome c550 from the diatom Phaeodactylum tricornutum suggest new alternative functions

Cytochrome c₅₅₀ is an extrinsic component in the luminal side of photosystem II (PSII) in cyanobacteria, as well as in eukaryotic algae from the red photosynthetic lineage including, among others, diatoms. We have established that cytochrome c₅₅₀ from the diatom Phaeodactylum tricornutum can be obtained as a complete protein from the membrane fraction of the alga, although a C‐terminal truncated form is purified from the soluble fractions of this diatom as well as from other eukaryotic algae. Eukaryotic cytochromes c₅₅₀ show distinctive electrostatic features as compared with cyanobacterial cytochrome c₅₅₀. In addition, co‐immunoseparation and mass spectrometry experiments, as well as immunoelectron microscopy analyses, indicate that although cytochrome c₅₅₀ from P. tricornutum is mainly located in the thylakoid domain of the chloroplast – where it interacts with PSII – , it can also be found in the chloroplast pyrenoid, related with proteins linked to the CO₂ concentrating mechanism and assimilation. These results thus suggest new alternative functions of this heme protein in eukaryotes.     

<Emphasis Type="Italic">Haloarcula marismortui</Emphasis> cytochrome <Emphasis Type="Italic">b-</Emphasis>561 is encoded by the <Emphasis Type="Italic">narC</Emphasis> gene in the dissimilatory nitrate reductase operon

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was −27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.     

Anti-Bcl-2 family members,zfBcl-x<Subscript>L</Subscript> and zfMcl-1a,prevent cytochrome <Emphasis Type="Italic">c</Emphasis> release from cells undergoing betanodavirus-induced secondary necrotic cell death

Nervous necrosis virus (NNV)-induced, host cell apoptosis mediates secondary necrosis by an ill-understood process. In this study, redspotted grouper nervous necrosis virus (RGNNV) is shown to induce mitochondria-mediated necrotic cell death in GL-av cells (fish cells) via cytochrome c release, and anti-apoptotic proteins are shown to protect these cells from death. Western blots revealed that cytochrome c release coincided with disruption of mitochondrial ultrastructure and preceded necrosis, but did not correlate with caspases activation. To identify the mediator(s) of this necrotic process, a protein synthesis inhibitor (cycloheximide; CHX; 0.33 μg/ml) was used to block cytochrome c release as well as PS exposure and mitochondrial membrane permeability transition pore (MMP) loss. CHX (0.33 μg/ml) completely blocked viral protein B2 expression, and partly blocked protein A, protein α, and a pro-apoptotic death protein (Bad) expression. Overexpression of B2 gene increased necrotic-like cell death up to 30% at 48 h post-transfection, suggesting that newly synthesized protein (B2) may be involved in this necrotic process. Finally, necrotic death was prevented by overexpression of Bcl-2 family proteins, zfBcl-x_L and xfMcl-1a. Thus, new protein synthesis and release of cytochrome c are required for RGNNV-induced necrotic cell death, which can be blocked by anti-apoptotic Bcl-2 members. J.-L. Wu and J.-R. Hong contributed equally to the research.     

Isolation of a cDNA encoding the human homolog of COX17, a yeast gene essential for mitochondrial copper recruitment

The COX17 gene of Saccharomyces cerevisiae codes for a cytoplasmic protein essential for the expression of functional cytochrome oxidase. This protein has been implicated in targeting copper to mitochondria. To determine if Cox17p is present in mammalian cells, a yeast strain carrying a null mutation in COX17 was transformed with a human cDNA expression library. All the respiratory competent clones obtained from the transformations carried a common cDNA sequence with a reading frame predicting a product homologous to yeast Cox17p. The cloning of a mammalian COX17 homolog suggests that the encoded product is likely to function in copper recruitment in eucaryotic cells in general. Its presence in humans provides a possible target for genetically inherited deficiencies in cytochrome oxidase. Received: 22 August 1996 / Revised: 31 October 1996     

Evolutionary divergence of the cytochrome b5 gene of Drosophila

Cytochrome proteins perform a broad spectrum of biological functions ranging from oxidative metabolism to electron transport and are thus essential to all organisms. The b-type cytochrome proteins bind heme noncovalently, are expressed in many different forms and are localized to various cellular compartments. We report the characterization of the cytochrome b₅ (Cyt-b) gene of Drosophila virilis and compare its structure to the Cyt-b gene of Drosophila melanogaster. As in D. melanogaster, the D. virilis gene is nuclear encoded and single copy. Although the intron/exon structures of these homologues differ, the Cyt-b proteins of D. melanogaster and D. virilis are approximately 75% identical and share the same size coding regions (1,242 nucleotides) and protein products (414 amino acids). The Drosophila Cyt-b proteins show sequence similarity to other b-type cytochromes, especially in the N-terminal heme-binding domain, and may be targeted to the mitochondrial membrane. The greatest levels of similarity are observed in areas of potential importance for protein structure and function. The exon sequences of the D. virilis Cyt-b gene differ by a total of 292 base changes. However, 62% of these changes are silent. The high degree of conservation between species separated by 60 million years of evolution in both the DNA and amino acid sequences suggests this nuclear cytochrome b₅ locus encodes an essential product of the Drosophila system.Correspondence to: C.E. Rozek     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.