A method for differentiating proteins from nucleic acids in intermediate-resolution density maps: cryo-electron microscopy defines the quaternary structure of the Escherichia coli 70S ribosome

BACKGROUND: This study addresses the general problem of dividing a density map of a nucleic-acid-protein complex obtained by cryo-electron microscopy (cryo-EM) or X-ray crystallography into its two components. When the resolution of the density map approaches approximately 3 A it is generally possible to interpret its shape (i. e., the envelope obtained for a standard choice of threshold) in terms of molecular structure, and assign protein and nucleic acid elements on the basis of their known sequences. The interpretation of low-resolution maps in terms of proteins and nucleic acid elements of known structure is of increasing importance in the study of large macromolecular complexes, but such analyses are difficult. RESULTS: Here we show that it is possible to separate proteins from nucleic acids in a cryo-EM density map, even at 11.5 A resolution. This is achieved by analysing the (continuous-valued) densities using the difference in scattering density between protein and nucleic acids, the contiguity constraints that the image of any nucleic acid molecule must obey, and the knowledge of the molecular volumes of all proteins. CONCLUSIONS: The new method, when applied to an 11.5 A cryo-EM map of the Escherichia coli 70S ribosome, reproduces boundary assignments between rRNA and proteins made from higher-resolution X-ray maps of the ribosomal subunits with a high degree of accuracy. Plausible predictions for the positions of as yet unassigned proteins and RNA components are also possible. One of the conclusions derived from this separation is that 23S rRNA is solely responsible for the catalysis of peptide bond formation. Application of the separation method to any nucleoprotein complex appears feasible.     

Partitioning of liquid-ordered/liquid-disordered membrane microdomains induced by the fluidifying effect of 2-hydroxylated fatty acid derivatives

Cellular functions are usually associated with the activity of proteins and nucleic acids. Recent studies have shown that lipids modulate the localization and activity of key membrane-associated signal transduction proteins, thus regulating the cell's physiology. Membrane Lipid Therapy aims to reverse cell dysfunctions (i.e., diseases) by modulating the activity of membrane signaling proteins through regulation of the lipid bilayer structure. The present work shows the ability of a series of 2-hydroxyfatty acid (2OHFA) derivatives, varying in the acyl chain length and degree of unsaturation, to regulate the membrane lipid structure. These molecules have shown greater therapeutic potential than their natural non-hydroxylated counterparts. We demonstrated that both 2OHFA and natural FAs induced reorganization of lipid domains in model membranes of POPC:SM:PE:Cho, modulating the liquid-ordered/liquid-disordered structures ratio and the microdomain lipid composition. Fluorescence spectroscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential detergent solubilization experiments showed a destabilization of the membranes upon addition of the 2OHFAs and FAs which correlated with the observed disordering effect. The changes produced by these synthetic fatty acids on the lipid structure may constitute part of their mechanism of action, leading to changes in the localization/activity of membrane proteins involved in signaling cascades, and therefore modulating cell responses.     

Binding of nucleic acids to intermediate filaments of the vimentin type and their effects on filament formation and stability.

Guanine-rich polynucleotides such as poly(dG), oligo(dG)12-18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)25 was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA, although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG).poly(dC), and, of those of the RNA type, preferentially poly(rA).poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

Binding of Nucleic Acids to Intermediate Filaments of the Vimentin Type and Their Effects on Filament Formation and Stability

Abstract

Guanine-rich polynucleotides such as poly(dG), oligo(dG)_12–18 or poly(rG) were shown to exert a strong inhibitory effect on vimentin filament assembly and also to cause disintegration of preformed filaments in vitro. Gold-labeled oligo(dG)₂₅ was preferentially localized at the physical ends of the aggregation and disaggregation products and at sites along filaments with a basic periodicity of 22.7 nm. Similar effects were observed with heat-denatured eukaryotic nuclear DNA or total rRNA although these nucleic acids could affect filament formation and structure only at ionic strengths lower than physiological. However, whenever filaments were formed or stayed intact, they appeared associated with the nucleic acids. These electron microscopic observations were corroborated by sucrose gradient analysis of complexes obtained from preformed vimentin filaments and radioactively labeled heteroduplexes. Among the duplexes of the DNA type, particularly poly(dG)·poly(dC), and, of those of the RNA type, preferentially poly(rA)·poly(rU), were carried by the filaments with high efficiency into the pellet fraction. Single-stranded 18S and 28S rRNA interacted only weakly with vimentin filaments. Nevertheless, in a mechanically undisturbed environment, vimentin filaments could be densely decorated with intact 40S and 60S ribosomal subunits as revealed by electron microscopy. These results indicate that, in contrast to single-stranded nucleic acids with their compact random coil configuration, double-stranded nucleic acids with their elongated and flexible shape have the capability to stably interact with the helically arranged, surface-exposed amino-terminal polypeptide chains of vimentin filaments. Such interactions might be of physiological relevance in regard to the transport and positioning of nucleic acids and nucleoprotein particles in the various compartments of eukaryotic cells. Conversely, nucleic acids might be capable of affecting the cytoplasmic organization of vimentin filament networks through their filament-destabilizing potentials.     

Solubilization of enzymes and nucleic acids in hydrocarbon micellar solutions

The present state of the field of biopolymers solubilized in apolar solvents via reverse micelles is reviewed. First, an extensive discussion of the physical and chemical properties of reverse micelles is presented. Particular attention is devoted to the nature of water in the water pools of reverse micelles; to the structure and shape of the micellar aggregates; and to the dynamic properties of the reverse micelles. In the second part of the paper, the mechanism of solubilization of proteins and nucleic acids in hydrocarbon reverse micelles is discussed. Spectroscopic data, mostly circular dichroism and fluorescence, are reviewed in order to clarify the conformational changes which the biopolymers undergo upon their uptake into the micellar environment and determine the location of the biopolymers inside the reverse micelles. Data from neutron scattering, light scattering, ultracentrifugation, and electron microscopy of the protein-containing micelles are reviewed and discussed with the aim of illustrating the structure of the micellar aggregates containing the biopolymer as guest molecules. The activity of enzymes and nucleic acids is discussed, with emphasis on the influence upon the chemical reactivity brought about by the micellar parameters. Finally, a brief review of the applications and potentialities of biopolymer-containing reverse micelles is presented.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena.

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Phosphoinositides,ezrin/moesin,and rac1 regulate fusion of rhodopsin transport carriers in retinal photoreceptors

The post-Golgi trafficking of rhodopsin in photoreceptor cells is mediated by rhodopsin-bearing transport carriers (RTCs) and regulated by the small GTPase rab8. In this work, we took a combined pharmacological-proteomic approach to uncover new regulators of RTC trafficking toward the specialized light-sensitive organelle, the rod outer segment (ROS). We perturbed phospholipid synthesis by activating phospholipase D with sphingosine 1-phosphate (S1P) or inhibiting phosphatidic acid phosphohydrolase by propranolol (Ppl). S1P stimulated the overall rate of membrane trafficking toward the ROS. Ppl stimulated budding of RTCs, but blocked membrane delivery to the ROS. Ppl caused accumulation of RTCs in the vicinity of the fusion sites, suggesting a defect in tethering, similar to the previously described phenotype of the rab8T22N mutant. Proteomic analysis of RTCs accumulated upon Ppl treatment showed a significant decrease in phosphatidylinositol-4,5-bisphosphate-binding proteins ezrin and/or moesin. Ppl induced redistribution of moesin, actin and the small GTPase rac1 from RTCs into the cytosol. By confocal microscopy, ezrin/moesin and rac1 colocalized with rab8 on RTCs at the sites of their fusion with the plasma membrane; however, this distribution was lost upon Ppl treatment. Our data suggest that in photoreceptors phosphatidylinositol-4,5-bisphosphate, moesin, actin, and rac1 act in concert with rab8 to regulate tethering and fusion of RTCs. Consequentially, they are necessary for rhodopsin-laden membrane delivery to the ROS, thus controlling the critical steps in the biogenesis of the light-detecting organelle.     

Characterization of alfalfa-mosaic-virus protein polymerization in the presence of nucleic acid

The polymerization of alfalfa mosaic virus (AMV) protein in the presence of homologous nucleic acids and a number of other natural and synthetic nucleic acids was studied. The conditions for optimal assembly were found to be pH 6.0 and low ionic strength (I = 0.1 M) at room temperature, irrespective of the type of nucleic acid. The resulting nucleoprotein particles exhibited the same structural characteristics as the virus. This information emerged from optical diffraction and computer filtering of electron micrographs from the reconstituted particles. Irrespective of the type of nucleic acid present the polymerization of the protein resulting in a nucleoprotein particle is a cooperative process. Evidence for this was obtained by nitrocellulose filter binding assay, sodium dodecylsulphate/polyacrylamide gel electrophoresis, sedimentation velocity and electron microscopy of the reaction mixtures. The rates and efficiencies of reconstitution were of the same order of magnitude for a number of ribonucleic acids. Sedimentation data derived from AMV protein and AMV RNA mixtures suggested the existence of a specific nucleation product in the first stage of assembly. The results are discussed in terms of a tentative model of the assembly, in which at least two different steps (nucleation and elongation) can be distinguished, each characterized by an association constant.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Study of early events during herpes simplex virus type 1 infection by confocal microscopy

Laser scanning confocal microscopy is a powerful technique that can be applied to study the localisation and behaviour of proteins and nucleic acids in many experimental situations. It is a particularly useful technique for the study of virus infections because of the changes that occur in the distribution and amounts of both viral and cellular proteins as infection develops. These changes reflect key stages and important regulatory events that govern the efficiency of infection. Using herpes simplex virus type 1 infected cells as an experimental model, this article provides guidance for users new to confocal microscopy on basic principles and techniques. The emphasis is on recognising, diagnosing and avoiding potential artifacts, and the workflow of the production of high quality, technically correct images.     

Structural characteristics of the desmin protofilament

Biochemical investigations of intermediate filaments in soluble or partially assembled forms are often difficult to perform due to the unusual insolubility of most types of intermediate filaments. However, desmin is soluble in 10 mM Tris. The structure of partially soluble native desmin was studied by gel-filtration chromatography and electron microscopy. The lowest molecular weight species of soluble desmin is a flexible rod averaging 53 nm in length. Calculations of f/fmin values from a previously published sedimentation value allowed comparisons with other elongated proteins. These values and the dimensions obtained from electron microscopy suggest that the desmin protofilament contains three or four protein subunits.