Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups.

Neuroglobin (Ngb) is a cytosolic heme protein that plays an important role in protecting cells from apoptosis through interaction with oxidized cytochrome c (Cyt c) released from mitochondria. The interaction of reduced Ngb and oxidized Cyt c is accompanied by electron transfer between them and the reduction in Cyt c. Despite the growing number of studies on Ngb, the mechanism of interaction between Ngb and Cyt c is still unclear. Using Raman spectroscopy, we studied the effect of charged amino acid substitutions in Ngb and Cyt c on the conformation of their hemes. It has been shown that Ngb mutants E60K, K67E, K95E and E60K/E87K demonstrate changed heme conformations with the lower probability of the heme planar conformation compared to wild-type Ngb. Moreover, oxidized Cyt c mutants K25E, K72E and K25E/K72E demonstrate the decrease in the probability of methyl-radicals vibrations, indicating the higher rigidity of the protein microenvironment. It is possible that these changes can affect electron transfer between Ngb and Cyt c.

Mutant Cytochrome C as a Potential Detector of Superoxide Generation: Effect of Mutations on the Function and Properties.

Cytochrome c (CytC) is a single-electron carrier between complex bc1 and cytochrome c-oxidase (CcO) in the electron transport chain (ETC). It is also known as a good radical scavenger but its participation in electron flow through the ETC makes it impossible to use CytC as a radical sensor. To solve this problem, a series of mutants were constructed with substitutions of Lys residues in the universal binding site (UBS) which interact electrostatically with negatively charged Asp and Glu residues at the binding sites of CytC partners, bc1 complex and CcO. The aim of this study was to select a mutant that had lost its function as an electron carrier in the ETC, retaining the structure and ability to quench radicals. It was shown that a mutant CytC with substitutions of five (8Mut) and four (5Mut) Lys residues in the UBS was almost inactive toward CcO. However, all mutant proteins kept their antioxidant activity sufficiently with respect to the superoxide radical. Mutations shifted the dipole moment of the CytC molecule due to seriously changed electrostatics on the surface of the protein. In addition, a decrease in the redox potential of the protein as revealed by the redox titrations of 8Mut was detected. Nevertheless, the CD spectrum and dynamic light scattering suggested no significant changes in the secondary structure or aggregation of the molecules of CytC 8Mut. Thus, a variant 8Mut with multiple mutations in the UBS which lost its ability to electron transfer and saved most of its physico-chemical properties can be effectively used as a detector of superoxide generation both in mitochondria and in other systems.

Molecular Interactions between Neuroglobin and Cytochrome c: Possible Mechanisms of Antiapoptotic Defense in Neuronal Cells.

Neuroglobin, which is a heme protein from the globin family that is predominantly expressed in nervous tissue, can promote a neuronal survivor. However, the molecular mechanisms underlying the neuroprotective function of Ngb remain poorly understood to this day. The interactions between neuroglobin and mitochondrial cytochrome c may serve as at least one of the mechanisms of neuroglobin-mediated neuroprotection. Interestingly, neuroglobin and cytochrome c possibly can interact with or without electron transfer both in the cytoplasm and within the mitochondria. This review provides a general picture of molecular interactions between neuroglobin and cytochrome c based on the recent experimental and computational work on neuroglobin and cytochrome c interactions.

Multiple Mutations in the Non-Ordered Red Ω-Loop Enhance the Membrane-Permeabilizing and Peroxidase-like Activity of Cytochrome c.

A key event in the cytochrome c-dependent apoptotic pathway is the permeabilization of the outer mitochondrial membrane, resulting in the release of various apoptogenic factors, including cytochrome c, into the cytosol. It is believed that the permeabilization of the outer mitochondrial membrane can be induced by the peroxidase activity of cytochrome c in a complex with cardiolipin. Using a number of mutant variants of cytochrome c, we showed that both substitutions of Lys residues from the universal binding site for oppositely charged Glu residues and mutations leading to a decrease in the conformational mobility of the red Ω-loop in almost all cases did not affect the ability of cytochrome c to bind to cardiolipin. At the same time, the peroxidase activity of all mutant variants in a complex with cardiolipin was three to five times higher than that of the wild type. A pronounced increase in the ability to permeabilize the lipid membrane in the presence of hydrogen peroxide, as measured by calcein leakage from liposomes, was observed only in the case of four substitutions in the red Ω-loop (M4 mutant). According to resonance and surface-enhanced Raman spectroscopy, the mutations caused significant changes in the heme of oxidized cytochrome c molecules resulting in an increased probability of the plane heme conformation and the enhancement of the rigidity of the protein surrounding the heme. The binding of wild-type and mutant forms of oxidized cytochrome c to cardiolipin-containing liposomes caused the disordering of the acyl lipid chains that was more pronounced for the M4 mutant. Our findings indicate that the Ω-loop is important for the pore formation in cardiolipin-containing membranes.

Kinetics and Energetics of Intramolecular Electron Transfer in Single-Point Labeled TUPS-Cytochrome c Derivatives.

Electron transfer within and between proteins is a fundamental biological phenomenon, in which efficiency depends on several physical parameters. We have engineered a number of horse heart cytochrome c single-point mutants with cysteine substitutions at various positions of the protein surface. To these cysteines, as well as to several native lysine side chains, the photoinduced redox label 8-thiouredopyrene-1,3,6-trisulfonate (TUPS) was covalently attached. The long-lived, low potential triplet excited state of TUPS, generated with high quantum efficiency, serves as an electron donor to the oxidized heme c. The rates of the forward (from the label to the heme) and the reverse (from the reduced heme back to the oxidized label) electron transfer reactions were obtained from multichannel and single wavelength flash photolysis absorption kinetic experiments. The electronic coupling term and the reorganization energy for electron transfer in this system were estimated from temperature-dependent experiments and compared with calculated parameters using the crystal and the solution NMR structure of the protein. These results together with the observation of multiexponential kinetics strongly support earlier conclusions that the flexible arm connecting TUPS to the protein allows several shortcut routes for the electron involving through space jumps between the label and the protein surface.

Lysine 72 substitutions differently affect lipid membrane permeabilizing and proapoptotic activities of horse heart cytochrome c.

Peroxidase activity of cytochrome c (cyt c)/cardiolipin (CL) complex is supposed to be involved in the initiation of apoptosis via peroxidative induction of mitochondrial membrane permeabilization. As cyt c binding to CL-containing membranes is at least partially associated with electrostatic protein/lipid interaction, we screened single-point mutants of horse heart cyt c with various substitutions of lysine at position 72, considered to play a significant role in both the binding and peroxidase activity of the protein. Contrary to expectations, K72A, K72R and K72L substitutions exerted slight effects on both the cyt c binding to CL-containing liposomal membranes and the cyt c/H2O2-induced calcein leakage from liposomes, used here as a membrane permeabilization assay. Both the binding and permeabilization were decreased to various extents, but not significantly, in the case of K72E and K72N mutants. A drastic difference was found between the sequence of the permeabilizing activities of the cyt c variants and the previously described order of their proapoptotic activities (Chertkova et al., 2008).

A Solid-State Protein Junction Serves as a Bias-Induced Current Switch.

A sample-type protein monolayer, that can be a stepping stone to practical devices, can behave as an electrically driven switch. This feat is achieved using a redox protein, cytochrome C (CytC), with its heme shielded from direct contact with the solid-state electrodes. Ab initio DFT calculations, carried out on the CytC-Au structure, show that the coupling of the heme, the origin of the protein frontier orbitals, to the electrodes is sufficiently weak to prevent Fermi level pinning. Thus, external bias can bring these orbitals in and out of resonance with the electrode. Using a cytochrome C mutant for direct S-Au bonding, approximately 80 % of the Au-CytC-Au junctions show at greater than 0.5 V bias a clear conductance peak, consistent with resonant tunneling. The on-off change persists up to room temperature, demonstrating reversible, bias-controlled switching of a protein ensemble, which, with its built-in redundancy, provides a realistic path to protein-based bioelectronics.

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells.

Structural and Dynamic "Portraits" of Recombinant and Native Cytotoxin I from Naja oxiana: How Close Are They?

Today, recombinant proteins are quite widely used in biomedical and biotechnological applications. At the same time, the question about their full equivalence to the native analogues remains unanswered. To gain additional insight into this problem, intimate atomistic details of a relatively simple protein, small and structurally rigid recombinant cardiotoxin I (CTI) from cobra Naja oxiana venom, were characterized using nuclear magnetic resonance (NMR) spectroscopy and atomistic molecular dynamics (MD) simulations in water. Compared to the natural protein, it contains an additional Met residue at the N-terminus. In this work, the NMR-derived spatial structure of uniformly 13C- and 15N-labeled CTI and its dynamic behavior were investigated and subjected to comparative analysis with the corresponding data for the native toxin. The differences were found in dihedral angles of only a single residue, adjacent to the N-terminal methionine. Microsecond-long MD traces of the toxins reveal an increased flexibility in the residues spatially close to the N-Met. As the detected structural and dynamic changes of the two CTI models do not result in substantial differences in their cytotoxicities, we assume that the recombinant protein can be used for many purposes as a reasonable surrogate of the native one. In addition, we discuss general features of the spatial organization of cytotoxins, implied by the results of the current combined NMR and MD study.

New insight into the mechanism of mitochondrial cytochrome c function.

We investigate functional role of the P76GTKMIFA83 fragment of the primary structure of cytochrome c. Based on the data obtained by the analysis of informational structure (ANIS), we propose a model of functioning of cytochrome c. According to this model, conformational rearrangements of the P76GTKMIFA83 loop fragment have a significant effect on conformational mobility of the heme. It is suggested that the conformational mobility of cytochrome c heme is responsible for its optimal orientation with respect to electron donor and acceptor within ubiquinol-cytochrome c oxidoreductase (complex III) and cytochrome c oxidase (complex IV), respectively, thus, ensuring electron transfer from complex III to complex IV. To validate the model, we design several mutant variants of horse cytochrome c with multiple substitutions of amino acid residues in the P76GTKMIFA83 sequence that reduce its ability to undergo conformational rearrangements. With this, we study the succinate-cytochrome c reductase and cytochrome c oxidase activities of rat liver mitoplasts in the presence of mutant variants of cytochrome c. The electron transport activity of the mutant variants decreases to different extent. Resonance Raman spectroscopy (RRS) and surface-enhanced Raman spectroscopy (SERS) data demonstrate, that all mutant cytochromes possess heme with the higher degree of ruffling deformation, than that of the wild-type (WT) cytochrome c. The increase in the ruffled deformation of the heme of oxidized cytochromes correlated with the decrease in the electron transport rate of ubiquinol-cytochrome c reductase (complex III). Besides, all mutant cytochromes have lower mobility of the pyrrol rings and methine bridges, than WT cytochrome c. We show that a decrease in electron transport activity in the mutant variants correlates with conformational changes and reduced mobility of heme porphyrin. This points to a significant role of the P76GTKMIFA83 fragment in the electron transport function of cytochrome c.

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms.

The fluorescent protein from Dendronephthya sp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C(α)-N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C(α)=C(ß) bond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Šresolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.

New strategy for high-level expression and purification of biologically active monomeric TGF-ß1/C77S in Escherichia coli.

Mature transforming growth factor beta1 (TGF-ß1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-ß1/C77S (further termed TGF-ß1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-ß1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-ß1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-ß1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-ß1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-ß1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-ß1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

Solid-state electron transport via cytochrome c depends on electronic coupling to electrodes and across the protein.

Electronic coupling to electrodes, Γ, as well as that across the examined molecules, H, is critical for solid-state electron transport (ETp) across proteins. Assessing the importance of each of these couplings helps to understand the mechanism of electron flow across molecules. We provide here experimental evidence for the importance of both couplings for solid-state ETp across the electron-mediating protein cytochrome c (CytC), measured in a monolayer configuration. Currents via CytC are temperature-independent between 30 and ∼130 K, consistent with tunneling by superexchange, and thermally activated at higher temperatures, ascribed to steady-state hopping. Covalent protein-electrode binding significantly increases Γ, as currents across CytC mutants, bound covalently to the electrode via a cysteine thiolate, are higher than those through electrostatically adsorbed CytC. Covalent binding also reduces the thermal activation energy, Ea, of the ETp by more than a factor of two. The importance of H was examined by using a series of seven CytC mutants with cysteine residues at different surface positions, yielding distinct electrode-protein(-heme) orientations and separation distances. We find that, in general, mutants with electrode-proximal heme have lower Ea values (from high-temperature data) and higher conductance at low temperatures (in the temperature-independent regime) than those with a distal heme. We conclude that ETp across these mutants depends on the distance between the heme group and the top or bottom electrode, rather than on the total separation distance between electrodes (protein width).

Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences.

Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) >> h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c approximately horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.