The role of syndecans in lymphoid systems

Syndecans, transmembrane heparan sulfate proteoglycans, play an important role in cell-cell and cell-matrix interactions, as receptors/co-receptors of matrix elements, cytokines, growth factors. These functions are partly non-specific and due to the heparan sulfate chains attached to the ectodomain, and partly specific related to the transmembrane and cytoplasmic domains of the core protein. In hemopoietic cells syndecan-1 is expressed in certain B cells, in pre-B cells and plasma cells. In lymphoproliferative diseases this normal syndecan-1 expression of plasma cells is retained in myelomas/plasmocytomas, other lymphoplasmocytic NHL subtypes and primary effusional lymphomas. Syndecan-1 expression is probably gained in B-CLL, and lost in other NHLs of pre- or post-follicular origin. These results suggest that the expression of syndecan is essential for some NHLs, probably ensuring the required connections to the microenvironment. From a diagnostic point of view, syndecan-1 is a very useful phenotypic marker to identify cells with plasmocytic differentiation. The importance of syndecan expression in CLL and Hodgkin-lymphoma still requires further studies.     

Role of the hinge protein in the electron transfer between cardiac cytochrome c1 and c. Equilibrium constants and kinetic probes

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.     

Four-dimensional microED of conformational dynamics in protein microcrystals on the femto-to-microsecond timescales

As structural determination of protein complexes approaches atomic resolution, there is an increasing focus on conformational dynamics. Here we conceptualize the combination of two techniques which have become established in recent years: microcrystal electron diffraction and ultrafast electron microscopy. We show that the extremely low dose of pulsed photoemission still enables microED due to the strength of the electron bunching from diffraction of the protein crystals. Indeed, ultrafast electron diffraction experiments on protein crystals have already been demonstrated to be effective in measuring intermolecular forces in protein microcrystals. We discuss difficulties that may arise in the acquisition and processing of data and the overall feasibility of the experiment, paying specific attention to dose and signal-to-noise ratio. In doing so, we outline a detailed workflow that may be effective in minimizing the dose on the specimen. A series of model systems that would be good candidates for initial experiments is provided.     

Amino Acid Sequences and Distribution of High-Potential Iron–Sulfur Proteins That Donate Electrons to the Photosynthetic Reaction Center in Phototropic Proteobacteria

High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2. For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2. We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2. We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.     

Monte Carlo calculations of energy deposition distributions of electrons below 20 keV in protein

The distributions of energy depositions of electrons in semi-infinite bulk protein and the radial dose distributions of point-isotropic mono-energetic electron sources [i.e., the so-called dose point kernel (DPK)] in protein have been systematically calculated in the energy range below 20 keV, based on Monte Carlo methods. The ranges of electrons have been evaluated by extrapolating two calculated distributions, respectively, and the evaluated ranges of electrons are compared with the electron mean path length in protein which has been calculated by using electron inelastic cross sections described in this work in the continuous-slowing-down approximation. It has been found that for a given energy, the electron mean path length is smaller than the electron range evaluated from DPK, but it is large compared to the electron range obtained from the energy deposition distributions of electrons in semi-infinite bulk protein. The energy dependences of the extrapolated electron ranges based on the two investigated distributions are given, respectively, in a power-law form. In addition, the DPK in protein has also been compared with that in liquid water. An evident difference between the two DPKs is observed. The calculations presented in this work may be useful in studies of radiation effects on proteins.     

Electron holography of non-stained bacterial surface layer proteins

We report transmission electron microscopy (TEM) investigations on bacterial surface layers (S-layers) which belong to the simplest biomembranes existing in nature. S-layers are regular 2D protein crystals composed of single protein or glycoprotein species. In their native form, S-layers are weak phase objects giving only poor contrast in conventional TEM. Therefore, they are usually examined negatively stained. However, staining with heavy metal compounds may cause the formation of structural artefacts. In this work, electron microscopy studies of non-stained S-layers of Bacillus sphaericus NCTC 9602 were performed. Compared to other proteins, these S-layers are found relatively stable against radiation damage. Electron holography was applied where information about phase and amplitude of the diffracted electron wave is simultaneously obtained. In spite of small phase shifts observed, the phase image reconstructed from the hologram of the non-stained S-layer is found to be sensitive to rather slight structure and thickness variations. The lateral resolution, obtained so far, is less than that of conventional electron microscopy of negatively stained S-layers. It corresponds to the main lattice planes of 12.4 nm observed in the reconstructed electron phase image. In addition, as a unique feature of electron holography the phase image provides thickness information. Thus, the existence of double layers of the protein crystals could be easily visualized by the height profile of the specimen.     

An engineered CuA Amicyanin capable of intermolecular electron transfer reactions

The type I copper center of amicyanin was replaced with a binuclear CuA center. To create this model CuA protein, a portion of the amino acid sequence that contains three of the ligands to the native type I copper center of Paracoccus denitrificans amicyanin was replaced with the corresponding portion of sequence that provides five ligands for the CuA center of cytochrome c oxidase from P. denitrificans. UV-visible and electron paramagnetic resonance spectroscopy confirm that the engineered protein as isolated possesses the mixed-valence Cu1.5Cu1.5 (purple) CuA center. Comparison of the spectroscopic properties of this CuA amicyanin with those of the CuA centers of other natural and engineered CuA proteins suggests that the spectroscopic features may be dictated more by the protein host than the sequence of the CuA loop. Novel reactions for a simple CuA model protein are also described. In contrast to other natural and engineered CuA proteins, the fully reduced CuA amicyanin may be reoxidized by molecular oxygen to the mixed-valence state. It is also shown that CuA amicyanin can serve as an electron donor and an electron acceptor for other redox proteins. The mixed-valence form accepts electrons from cytochromes c-551i and c-550 from P. denitrificans. The fully reduced form donates electrons to native and P94F amicyanin. The function as either an electron donor or acceptor is consistent with the measured redox potential of CuA amicyanin of +273 mV. These data indicate that this CuA amicyanin will be a particularly useful model protein for structure-function studies of reactivity and the electron transfer properties of the CuA redox center.     

Lipid/myelin basic protein multilayers. A model for the cytoplasmic space in central nervous system myelin

A multilayered complex forms when a solution of myelin basic protein is added to single-bilayer vesicles formed by sonicating myelin lipids. Vesicles and multilayers have been studied by electron microscopy, biochemical analysis, and X-ray diffraction. Freeze-fracture electron microscopy shows well-separated vesicles before myelin basic protein is added, but afterward there are aggregated, possibly multilayered, vesicles and extensive planar multilayers. The vesicles aggregate and fuse within seconds after the protein is added, and the multilayers form within minutes. No intra-bilayer particles are seen, with or without the protein. Some myelin basic protein, but no lipid, remains in the supernatant after the protein is added and the complex sedimented for X-ray diffraction. A rather variable proportion of the protein is bound. X-ray diffraction patterns show that the vesicles are stable in the absence of myelin basic protein, even under high g-forces. After the protein is added, however, lipid/myelin basic protein multilayers predominate over single-bilayer vesicles. The protein is in every space between lipid bilayers. Thus the vesicles are torn open by strong interaction with myelin basic protein. The inter-bilayer spaces in the multilayers are comparable to the cytoplasmic spaces in central nervous system myelins . The diffraction indicates the same lipid bilayer thickness in vesicles and multilayers, to within 1 A. By comparing electron-density profiles of vesicles and multilayers, most of the myelin basic protein is located in the inter-bilayer space while up to one-third may be inserted between lipid headgroups. When cytochrome c is added in place of myelin basic protein, multilayers also form. In this case the protein is located entirely outside the unchanged bilayer. Comparison of the various profiles emphasizes the close and extensive apposition of myelin basic protein to the lipid bilayer. Numerous bonds may form between myelin basic protein and lipids. Cholesterol may enhance binding by opening gaps between diacyl-lipid headgroups.     

Electron spectroscopic imaging of chromatin.

The analytical electron microscope technique called electron spectroscopic imaging (ESI) has a number of applications in the study of DNA:protein complexes. The method offers an intermediate level of spatial resolution for in vitro structural studies of complexes that may be too large or heterogeneous to study by crystallography or magnetic resonance spectroscopy. An advantage of ESI is that the distribution of nucleic acids can be resolved in a nucleoprotein complex by mapping the element phosphorus, present at high levels in nucleic acid compared to protein. Measurements of phosphorus content together with mass determination allows estimates to be made of stoichiometric relationships of protein and nucleic acids in these complexes. ESI is also suited to in situ studies of nuclear structure. Mass-sensitive images combined with nitrogen and phosphorus maps can be used to distinguish nucleic acid components from nuclear structures that are predominantly protein based. Interactions between chromatin on the periphery of interchromatin granule clusters (IGC) with the protein substructure that connects the exterior of the IGC to its core can be studied with this technique. The method also avoids the use of heavy atom stains, agents required in conventional electron microscopy, that preclude the distinguishing of structures on the basis of their biochemical composition. The principles of ESI and technical aspects of the method are discussed.     

Studies on intercellular LETS glycoprotein matrices

Intercellular matrices secreted by chick embryo fibroblasts in culture were studied by scanning electron microscopy. Cell-cell contact is a prerequisite for the expression of such matrices. The smallest fiber detected by transmission electron microscopy is 5--10 nm in diameter. These matrix fibers tend to cluster to form bundles. Immunofluorescence and immunoferritin procedures reveal that LETS protein is one of the components of the matrices. The matrices are isolated from other cellular organelles by detergent treatment. More than 90% of the proteins in cell-free matrices are LETS protein, suggesting that the matrices are probably made of only one component--LETS protein. Since the solubilization of matrices requires beta-mercaptoethanol, LETS protein matrices may be the first known polymer system in nature to use disulfide linkage as an intermolecular polymerization vehicle. Collagen does not appear to be involved in such matrices. The LETS protein matrix supports the morphological conversion of rounded cells into spindle-shaped, and also promotes myoblast fusion. It does not, however, exert an effect upon cell growth, the rate of glucose uptake or protease production.     

Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 A resolution

Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 A resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and pi-pi interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H...SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium.     

Structural analysis of membrane protein complexes by single particle electron microscopy

Single particle electron microscopy (EM) is an increasingly important tool for the structural analysis of macromolecular complexes. The main advantage of the technique over other methods is that it is not necessary to precede the analysis with the growth of crystals of the sample. This advantage is particularly important for membrane proteins and large protein complexes where generating crystals is often the main barrier to structure determination. Therefore, single particle EM can be employed with great utility in the study of large membrane protein complexes. Although the construction of atomic resolution models by single particle EM is possible in theory, currently the highest resolution maps are still limited to approximately 7-10A resolution and 15-30 A resolution is more typical. However, by combining single particle EM maps with high-resolution models of subunits or subcomplexes from X-ray crystallography and NMR spectroscopy it is possible to build up an atomic model of a macromolecular assembly. Image analysis procedures are almost identical for micrographs of soluble protein complexes and detergent solubilized membrane protein complexes. However, electron microscopists attempting to prepare specimens of a membrane protein complex for imaging may find that these complexes require different handling than soluble protein complexes. This paper seeks to explain how high-quality specimen grids of membrane protein complexes may be prepared to allow for the determination of their structure by EM and image analysis.     

Lipid fluidity and membrane protein dynamics

Membrane fluidity plays an important role in cellular functions. Membrane proteins are mobile in the lipid fluid environment; lateral diffusion of membrane proteins is slower than expected by theory, due to both the effect of protein crowding in the membrane and to constraints from the aqueous matrix. A major aspect of diffusion is in macromolecular associations: reduction of dimensionality for membrane diffusion facilitates collisional encounters, as those concerned with receptor-mediated signal transduction and with electron transfer chains. In mitochondrial electron transfer, diffusional control is prevented by the excess of collisional encounters between fast-diffusing ubiquinone and the respiratory complexes. Another aspect of dynamics of membrane proteins is their conformational flexibility. Lipids may induce the optimal conformation for catalytic activity. Breaks in Arrhenius plots of membrane-bound enzymes may be related to lipid fluidity: the break could occur when a limiting viscosity is reached for catalytic activity. Viscosity can affect protein conformational changes by inhibiting thermal fluctuations to the inner core of the protein molecule.     

A probabilistic approach to protein backbone tracing in electron density maps

One particularly time-consuming step in protein crystallography is interpreting the electron density map; that is, fitting a complete molecular model of the protein into a 3D image of the protein produced by the crystallographic process. In poor-quality electron density maps, the interpretation may require a significant amount of a crystallographer's time. Our work investigates automating the time-consuming initial backbone trace in poor-quality density maps. We describe ACMI (Automatic Crystallographic Map Interpreter), which uses a probabilistic model known as a Markov field to represent the protein. Residues of the protein are modeled as nodes in a graph, while edges model pairwise structural interactions. Modeling the protein in this manner allows the model to be flexible, considering an almost infinite number of possible conformations, while rejecting any that are physically impossible. Using an efficient algorithm for approximate inference--belief propagation--allows the most probable trace of the protein's backbone through the density map to be determined. We test ACMI on a set of ten protein density maps (at 2.5 to 4.0 A resolution), and compare our results to alternative approaches. At these resolutions, ACMI offers a more accurate backbone trace than current approaches.     

Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.     

Protein bodies in seeds

Membrane bound protein bodies (aleurone grains) are thought to be the main subcellular location of protein and mineral storage in seeds. In addition to structurally homogeneous proteinaceous matrix, protein bodies may contain protein crystalloids, electron–dense globoid crystals, electron–transparent soft globoids, and crystals of calcium oxalate. Protein crystalloids vary in shape, size and number. For example, cotyledon mesophyll cell protein bodies in the Cucurbitaceae generally contain protein crystalloids whereas those of Compositae and Cruciferae do not. Globoid crystals, which are rich in phytin, vary greatly in size and number per protein body. Some species have numerous small globoid crystals per protein body whereas others have one or two large globoid crystals per protein body. Phosphorus and various cations (K, Mg, Ca, Fe, Ba, Mn) located in globoid crystals can be studied with an energy dispersive X–ray (EDX) analysis system mounted on a transmission electron microscope. In some cases, cations such as Ca, Mn and Fe are specifically localized in globoid crystals of certain tissues or embryo regions. Further investigations may allow elemental composition of globoid crystals to be used in studies of systematics. Biref–ringent crystals, sometimes in the form of single large crystals but frequently in the form of druses, are present in protein bodies of some species. At least some endosperm protein bodies of all Umbelliferous species examined contain druse crystals. While seed protein bodies of relatively few species have been studied with electron microscopy, there are indications that protein bodies could be a useful character for studies in plant systematics.     

Are mitochondria a spontaneous and permanent source of reactive oxygen species?

The bioenergetic properties of mitochondria in combination with the high turnover rate of dioxygen qualify these organelles for the formation of reactive oxygen species (ROS). The assumption that mitochondria are the major intracellular source of ROS was essentially based on in vitro experiments with isolated mitochondria. The transfer of these data to the living cell may, however, be incorrect. Artefacts due to the preparation procedure or inadequate detection methods of ROS may lead to false positive results. Inhomogeneous results were found to be due to an interaction of the detection system with components of the respiratory chain which could be avoided by a recently developed non-invasive method. One of the most critical electron transfer steps in the respiratory chain is the electron bifurcation from ubiquinol to the cytochrome bc(1) complex. This electron bifurcation requires the free mobility of the head domain of the Rieske iron-sulfur protein. Inhibition of electron bifurcation by antimycin A causes leakage of single electrons to oxygen which results in the release of ROS. Hindrance of electron bifurcation was also observed following alterations of the physical state of membrane phospholipids in which the cytochrome bc(1) complex is inserted. Irrespective of whether the fluidity of the membrane was elevated or decreased, electron flow rates to the Rieske iron-sulfur protein were drastically reduced. Concomitantly superoxide radicals were released from these mitochondria, strongly suggesting the involvement of the ubiquinol/cytochrome bc(1) redox couple in this process.