Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.     

Proteins of the cyanobacterial photosystem I.

Cyanobacterial photosystem (PS) I is remarkably similar to its counterpart in the chloroplast of plants and algae. Therefore, it has served as a prototype for the type I reaction centers of photosynthesis. Cyanobacterial PS I contains 11-12 proteins. Some of the cyanobacterial proteins are modified post-translationally. Reverse genetics has been used to generate subunit-deficient cyanobacterial mutants, phenotypes of which have revealed the functions of the missing proteins. The cyanobacterial PS I proteins bind cofactors, provide docking sites for electron transfer proteins, participate in tertiary and quaternary organization of the complex and protect the electron transfer centers. Many of these mutants are now being used in sophisticated structure-function analyses. Yet, the roles of some proteins of the cyanobacterial PS I are unknown. It is necessary to examine functions of these proteins on a global scale of cell physiology, biogenesis and evolution.     

Translocation of DNA across bacterial membranes.

DNA translocation across bacterial membranes occurs during the biological processes of infection by bacteriophages, conjugative DNA transfer of plasmids, T-DNA transfer, and genetic transformation. The mechanism of DNA translocation in these systems is not fully understood, but during the last few years extensive data about genes and gene products involved in the translocation processes have accumulated. One reason for the increasing interest in this topic is the discussion about horizontal gene transfer and transkingdom sex. Analyses of genes and gene products involved in DNA transfer suggest that DNA is transferred through a protein channel spanning the bacterial envelope. No common model exists for DNA translocation during phage infection. Perhaps various mechanisms are necessary as a result of the different morphologies of bacteriophages. The DNA translocation processes during conjugation, T-DNA transfer, and transformation are more consistent and may even be compared to the excretion of some proteins. On the basis of analogies and homologies between the proteins involved in DNA translocation and protein secretion, a common basic model for these processes is presented.     

Long range electron transfer along an alpha-helix.

The many observations of long range electron transfer in proteins raises the question of whether a protein's structure can influence the rate or path of such transfers, and if so, then how. To answer these questions requires information on which of the various structural elements composing proteins support long range electron transfer. In this report, we present evidence for long range electron transfer along the alpha-helix of a synthetic leucine zipper dimer. We also present electron transfer rate data obtained with other helical peptides.     

Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes.

Using elicitins, proteins secreted by some phytopathogenic Oomycetes (Phytophthora) known to be able to transfer sterols between phospholipid vesicles, the transfer of sterols between micelles, liposomes and biological membranes was studied. Firstly, a simple fluorometric method to screen the sterol-carrier capacity of proteins, avoiding the preparation of sterol-containing phospholipidic vesicles, is proposed. The transfer of sterols between DHE micelles (donor) and stigmasterol or cholesterol micelles (acceptor) was directly measured, as the increase in DHE fluorescence signal. The results obtained with this rapid and easy method lead to the same conclusions as those previously reported, using fluorescence polarization of a mixture of donor and acceptor phospholipid vesicles, prepared in the presence of different sterols. Therefore, the micelles method can be useful to screen proteins for their sterol carrier activity. Secondly, elicitins are shown to trap sterols from purified plant plasma membranes and to transfer sterols from micelles to these biological membranes. This property should contribute to understand the molecular mechanism involved in sterol uptake by Phytophthora. It opens new perspectives concerning the role of such proteins in plant-microorganism interactions.     

Western blots using stained protein gels.

A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.     

Genealogy of legume-Rhizobium symbioses.

Accumulating evidence suggests that lateral transfer of nodulation capacity is an important driving force in symbiotic evolution. As a consequence, many distantly related soil bacteria have acquired the capacity to invade plants and fix nitrogen within them. In addition to these proteins required for bacteroid development and nitrogen fixation, core symbiotic competence seems to require flavonoids, NodD proteins, lipochitooligosaccharidic Nod-factors, extra-cellular polysaccharides, as well as various exported proteins. Plants respond to different levels and combinations of these substances in species specific ways. After contact has been initiated by flavonoids and NodD proteins, constant signal exchange fine-tunes these symbiotic demands, especially to overcome defence reactions.     

Determination of the fractal dimension of membrane protein aggregates using fluorescence energy transfer.

It is demonstrated that fluorescence resonance energy transfer may be used to determine the fractal dimension of aggregates of membrane-bound proteins. Theoretical and experimental results are presented for two different experimental designs: energy transfer between proteins and energy transfer from lipids to proteins. For energy transfer between proteins the lattice spacing must be known independently for a fractal dimension to be uniquely determined, and this represents a disadvantage to this experimental design. Results are presented for the calcium ATPase and a fractal dimension of 1.9 is estimated for ATPase aggregates by assuming a lattice spacing of 50 A. Energy transfer from lipids to protein provides a means of estimating the length of the "coast-line" of the aggregate. In this case the fractal dimension is uniquely determined from a log-log plot. An analysis of data for bacteriohodopsin reconstituted in phospholipid vesicles gives a fractal dimension of 1.6. The structural basis of the value for the fractal dimension is discussed for these two systems. These techniques provide a means of assessing the nature of protein-protein interactions in membranous systems.     

Role of the helical domain in fatty acid transfer from adipocyte and heart fatty acid-binding proteins to membranes: analysis of chimeric proteins.

The adipocyte and heart fatty acid-binding proteins (A- and HFABP) are members of a lipid-binding protein family with a beta-barrel body capped by a small helix-turn-helix motif. Both proteins are hypothesized to transport fatty acid (FA) to phospholipid membranes through a collisional process. Previously, we suggested that the helical domain is particularly important for the electrostatic interactions involved in this transfer mechanism (Herr, F. M., Aronson, J., and Storch, J. (1996) Biochemistry 35, 1296-1303; and Liou, H.-L., and Storch, J. (2001) Biochemistry 40, 6475-6485). Despite their using qualitatively similar FA transfer mechanisms, differences in absolute transfer rates as well as regulation of transfer from AFABP versus HFABP, prompted us to consider the structural determinants that underlie these functional disparities. To determine the specific elements underlying the functional differences between AFABP and HFABP in FA transfer, two pairs of chimeric proteins were generated. The first and second pairs had the entire helical domain and the first alpha-helix exchanged between A- and HFABP, respectively. The transfer rates of anthroyloxy-labeled fatty acid from proteins to small unilamellar vesicles were compared with the wild type AFABP and HFABP. The results suggest that the alphaII-helix is important in determining the absolute FA transfer rates. Furthermore, the alphaI-helix appears to be particularly important in regulating protein sensitivity to the negative charge of membranes. The alphaI-helix of HFABP and the alphaII-helix of AFABP increased the sensitivity to anionic vesicles; the alphaI-helix of AFABP and alphaII-helix of HFABP decreased the sensitivity. The differential sensitivities to negative charge, as well as differential absolute rates of FA transfer, may help these two proteins to function uniquely in their respective cell types.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron-sulfur cluster biosynthesis. Kinetic analysis of [2Fe-2S] cluster transfer from holo ISU to apo Fd: role of redox chemistry and a conserved aspartate

ISU-type proteins mediate cluster transfer to apo protein targets. Rate constants have been determined for cluster transfer from ISU to apo Fd for both Homo sapiens and Schizosaccharomyces pombe proteins, and cross reactions have also been examined. Substitution of a key aspartate residue of ISU is found to decrease the rate of cluster transfer by at least an order of magnitude (for wild-type Hs ISU cluster transfer to Hs apo Fd, k(2) approximately 540 M(-1) min(-1), relative 56 M(-1) min(-1) for D37A ISU). This change in rate constant does not reflect any change in binding affinity of the ISU and Fd proteins. The pH dependencies of cluster transfer rates are similar for WT and D37A ISU, arguing against a role for Asp37 as a catalytic base, although evidence for general base catalysis mediating deprotonation of Cys from the apo target is supported by an observed pK(a) of 6.9 determined from the pH profiles for both WT and D37A ISU. Such a pK(a) value is at the lower limit for Cys and is common for solvent-accessible Cys thiols. The temperature dependence of the rate constant defining the cluster transfer reaction for wild type versus the aspartate derivative is distinct. Thermal activation parameters (DeltaH and DeltaS) are consistent with a solvent-accessible ISU-bound cluster, with desolvation as a principle barrier to cluster transfer. Experiments to determine the dependence of reaction rate constants on viscosity indicate cluster transfer to be rate-limiting. Fully oxidized cluster appears to be the natural state for transfer to target proteins. Reduced Fd does not readily reduce ISU-bound [2Fe-2S](2+) and does not promote cluster transfer to an apo Fd target.     

Free fatty acid transfer from rat liver fatty acid-binding protein to phospholipid vesicles. Effect of ligand and solution properties.

Fatty acid binding proteins (FABP) are a family of 14-15-kDa proteins found in many mammalian cell types in high abundance. Although their precise physiological role remains hypothetical, the transfer of free fatty acids (ffa) to intracellular membrane sites is believed to be an important function of FABP. To better understand the role of FABP in this process, we have examined how the rate of ffa transfer from liver FABP (L-FABP) to model membranes is influenced by variations in ffa structure and properties of the aqueous phase. The rate of transfer of fluorescent anthroyloxy ffa to model acceptor membranes was monitored using a resonance energy transfer assay. The results show that a monounsaturated ffa transfers 2-fold more rapidly than a saturated ffa of equivalent chain length, and a two-carbon increase in acyl chain length results in a 3-fold decrease in transfer rate. The transfer rate decreases logarithmically with increasing ionic strength, suggesting that the aqueous solubility of the ffa is an important determinant of its dissociation rate from L-FABP. Fatty acid binding and the relative partition of n-(9-anthroloxy) ffa to L-FABP as compared with phospholipid membranes both decrease as pH decreases, indicating that ionized but not protonated ffa bind to L-FABP. The rate of ffa transfer from L-FABP to membranes increases approximately 4-fold with increasing pH, suggesting that ionization of the ffa carboxyl group is also an important determinant of the transfer process. Analysis of the dependence of the transfer rate on temperature demonstrates that the delta G++ of the activated state for ffa transfer arises from both enthalpic and entropic processes. These studies demonstrate that the rate of transfer of long chain ffa from L-FABP to membranes is substantially affected by aqueous phase variables as well as properties of the ffa ligand itself.     

Transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride membranes.

We have developed a method to transfer proteins from a silver-stained polyacrylamide gel to a polyvinylidene difluoride (Immobilon-P) transfer membrane (Millipore, Bedford, MA). If the silver stained gels are rinsed in 2 x SDS Laemmli sample buffer prior to transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transfer, almost all proteins can be transferred comparably to non-stained controls. Some proteins stained with silver can be directly transferred to a single sheet of Immobilon-P without a prior rinse in sample buffer. Most important in the Western blot the antigenicity of the transferred protein is retained in either way. The method described is simple, inexpensive and versatile. A slight modification of the technique permits one to extract minor proteins, or detect their antigenic activities, without contamination of contiguous proteins.     

The redox potentials of the two-iron plant algal ferredoxins. An electrostatic model

The two-iron-sulphur co-ordination centre in plant and algal ferredoxins is considered as a collection of charged ions whose net negative charge is twice that of the one-iron-sulphur protein rubredoxin. Calculation of the electrostatic free-energy changes for reduction of the two types of proteins indicates that the redox potential of the two-iron-sulphur proteins should be more negative than that of the one-iron-sulphur protein and that in biological systems the ferredoxins should function as one-electron transfer proteins.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.     

Solid-state protein kinase. A tool for post-synthetically modifying and radioactively labeling proteins in vitro.

The capacity of partially purified rat muscle protein kinase coupled to cyanogen-bromide-activated Sepharose 4B to (radio-)phosphorylate proteins in vitro was evaluated using histones from calf thymus and rat liver and certain proteins as substrates. Data are presented which point to a low substrate specificity of this enzyme. It is demonstrated that even within a short time period histones are efficiently phosphorylated without the introduction of contaminating (phospho-)proteins. Therebye phosphoserine residues are formed. The phosphorylation reaction usually performed at 30 degrees C is shown to function quite efficiently also at 4 degrees C. It proceeds even at 30 degrees C for several hours at pH values close to the physiological range without the release of proteins from the solid matrix. The phosphorus transfer can be largely increased with the use of high ATP concentrations. The stability of the substrates is sufficient to suggest a wide applicability of this solid-state protein kinase in the phosphorylation of proteins either for labeling or as a tool to modify proteins post-synthetically under gentle conditions. The solid enzyme seems to be suitable for radioactively labeling proteins of more complex biological structures, such as membrane surfaces.     

Stimulated and co-operative electron transfer in energy conversion and catalysis.

A new mechanism of electron transfer, stimulated electron transfer, is postulated, in which an electronic feedback is drastically increasing both the rate of electron transfer and the propagation of free energy along electron transferring molecular pathways. In principle, the idea of pushing a system far from equilibrium to achieve a high reaction rate and co-operative phenomena is applied to molecular electron transfer. The effect is calculated from a semiclassical kinetic model of a chain redox reaction with autocatalytic feedback on individual rate constants, where the steps have subsequently been minimized to obtain a continuous electron transfer pathway with electronic feedback. The influence of inhomogeneities and asymmetries in the electron transfer path and of vectorial components (electrical field, gradient of redox potential) are discussed as well as the acceleration of individual and multiple electron transfer as a function of feedback. Examples of autocatalytic feedback are provided including mechanisms involving electron transfer proteins and multi-centre electron transfer catalysts. Such a phenomenon can be described for molecular and interfacial electron transfer in analogy to stimulated and coherent light emission. The results suggest that autocatalytic or stimulated electron transfer may be a key to the understanding of efficient electron transfer and co-operative multi-electron transfer catalysis in biology and a challenge for fuel production mechanisms in artificial photosynthesis and fuel cycles.     

Insights into the design of a hybrid system between Anabaena ferredoxin-NADP+ reductase and bovine adrenodoxin.

The opportunity to design enzymatic systems is becoming more feasible due to detailed knowledge of the structure of many proteins. As a first step, investigations have aimed to redesign already existing systems, so that they can perform a function different from the one for which they were synthesized. We have investigated the interaction of electron transfer proteins from different systems in order to check the possibility of heterologous reconstitution among members of different chains. Here, it is shown that ferredoxin-NADP+ reductase from Anabaena and adrenodoxin from bovine adrenal glands are able to form optimal complexes for thermodynamically favoured electron transfer reactions. Thus, electron transfer from ferredoxin-NADP+ reductase to adrenodoxin seems to proceed through the formation of at least two different complexes, whereas electron transfer from adrenodoxin to ferredoxin-NADP+ reductase does not take place due because it is a thermodynamically nonfavoured process. Moreover, by using a truncated adrenodoxin form (with decreased reduction potential as compared with the wild-type) ferredoxin-NADP+ reductase is reduced. Finally, these reactions have also been studied using several ferredoxin-NADP+ reductase mutants at positions crucial for interaction with its physiological partner, ferredoxin. The effects observed in their reactions with adrenodoxin do not correlate with those reported for their reactions with ferredoxin. In summary, our data indicate that although electron transfer can be achieved in this hybrid system, the electron transfer processes observed are much slower than within the physiological partners, pointing to a low specificity in the interaction surfaces of the proteins in the hybrid complexes.     

Comparative genomic analyses of the bacterial phosphotransferase system.

We report analyses of 202 fully sequenced genomes for homologues of known protein constituents of the bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS). These included 174 bacterial, 19 archaeal, and 9 eukaryotic genomes. Homologues of PTS proteins were not identified in archaea or eukaryotes, showing that the horizontal transfer of genes encoding PTS proteins has not occurred between the three domains of life. Of the 174 bacterial genomes (136 bacterial species) analyzed, 30 diverse species have no PTS homologues, and 29 species have cytoplasmic PTS phosphoryl transfer protein homologues but lack recognizable PTS permeases. These soluble homologues presumably function in regulation. The remaining 77 species possess all PTS proteins required for the transport and phosphorylation of at least one sugar via the PTS. Up to 3.2% of the genes in a bacterium encode PTS proteins. These homologues were analyzed for family association, range of protein types, domain organization, and organismal distribution. Different strains of a single bacterial species often possess strikingly different complements of PTS proteins. Types of PTS protein domain fusions were analyzed, showing that certain types of domain fusions are common, while others are rare or prohibited. Select PTS proteins were analyzed from different phylogenetic standpoints, showing that PTS protein phylogeny often differs from organismal phylogeny. The results document the frequent gain and loss of PTS protein-encoding genes and suggest that the lateral transfer of these genes within the bacterial domain has played an important role in bacterial evolution. Our studies provide insight into the development of complex multicomponent enzyme systems and lead to predictions regarding the types of protein-protein interactions that promote efficient PTS-mediated phosphoryl transfer.     

Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts.

Double-stranded DNA (dsDNA) induces the transfer of phosphate from ATP to several proteins in extracts of widely divergent eukaryotic cells. Extracts of HeLa cells, rabbit reticulocytes, Xenopus eggs and Arbacia eggs all show dsDNA-dependent protein phosphorylation. The mechanism is specific for dsDNA and will not respond to either RNA or single-stranded DNA. One of the proteins which is phosphorylated in response to dsDNA has a subunit mol. wt. of 90 000 and has been identified as a heat-shock protein (hsp90). Although mouse cell extracts were shown to contain hsp90, they failed to show a dsDNA-dependent protein phosphorylation. The observation that dsDNA can modulate the phosphorylation of a set of proteins raises the possibility that dsDNA may play a role as a cellular regulatory signal.