Mechanical and structural properties of in vitro neurofilament hydrogels

Neurofilaments belong to the class of cytoskeletal intermediate filaments and are the predominant structural elements in axons. They are composed of a semiflexible backbone and highly charged anionic sidearms protruding from the surface of the filaments. Here, the rheology of in-vitro networks of neurofilaments purified from pig spinal cord was determined. The mechanical properties of these networks are qualitatively similar to other hydrogels of semiflexible polymers. The low-deformation storage modulus G'(omega) showed a concentration (c) dependence of G' approximately c (1.3) that is consistent with a model for semiflexible networks, but was also observed for polyelectrolyte brushes. A terminal relaxation was not observed in the frequency range investigated (0.007-5 Hz), supporting the notion that sidearms act as cross-links hindering slip between filaments on a time scale of many minutes. The mesh size distribution of the network was measured by analysis of Brownian motion of embedded beads. The concentration dependence of the mesh size follows the same power law behaviour as found for F-actin networks, but shows a significantly wider distribution attributable to the smaller persistence length of neurofilaments. The attractive interaction between filaments is increased by addition of Al(3+) ions resulting in a reduction of the linear response regime from strains bigger than 80% to less than 30%.     

Mechanical and structural properties of YOYO-1 complexed DNA

YOYO-1 is a fluorescent dye widely used for probing the statistical–mechanical properties of DNA. However, currently contradicting data exist how YOYO-1 binding alters the DNA structure and rigidity. Here, we systematically address this problem using magnetic tweezers. Remarkably, we find that the persistence length of DNA remains constant independent of the amount of bound YOYO-1, which contrasts previous assumptions. While the ionic conditions can considerably alter the stability of YOYO-1 binding, the DNA bending rigidity seems not to be affected. We furthermore determine important structural parameters such as the binding site size, the elongation, as well as the untwisting angle per bound YOYO-1 molecule. We expect that our assay, in which all the parameters are determined within a single experiment, will be beneficial for a large range of other DNA binding drugs.     

Mechanical deformation behaviors and structural properties of ligated DNA crystals

DNA self-assembly has emerged as a powerful strategy for constructing complex nanostructures. While the mechanics of individual DNA strands have been studied extensively, the deformation behaviors and structural properties of self-assembled architectures are not well understood. This is partly due to the small dimensions and limited experimental methods available. DNA crystals are macroscopic crystalline structures assembled from nanoscale motifs via sticky-end association. The large DNA constructs may thus be an ideal platform to study structural mechanics. Here, we investigate the fundamental mechanical properties and behaviors of ligated DNA crystals made of tensegrity triangular motifs. We perform coarse-grained molecular dynamics simulations and confirm the results with nanoindentation experiments using atomic force microscopy. We observe various deformation modes, including untension, linear elasticity, duplex dissociation, and single-stranded component stretch. We find that the mechanical properties of a DNA architecture are correlated with those of its components. However, the structure shows complex behaviors which may not be predicted by components alone and the architectural design must be considered.     

Enzymatic and structural characterization of an archaeal thiamin phosphate synthase

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme–product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.     

Crystal structures of two archaeal Pelotas reveal inter-domain structural plasticity

Dom34 from Saccharomyces cerevisiae is one of the key players in no-go mRNA decay, a surveillance pathway by which an abnormal mRNA stalled during translation is degraded by an endonucleolytic cleavage. Its homologs called Pelota are found in other species. We showed previously that S. cerevisiae Dom34 (domain 1) has an endoribonuclease activity, which suggests its direct catalytic role in no-go decay. Pelota from Thermoplasma acidophilum and Dom34 from S. cerevisiae have been structurally characterized, revealing a tripartite architecture with a significant difference in their overall conformations. To gain further insights into structural plasticity of the Pelota proteins, we have determined the crystal structures of two archaeal Pelotas from Archaeoglobus fulgidus and Sulfolobus solfataricus. Despite the structural similarity of their individual domains to those of T. acidophilum Pelota and S. cerevisiae Dom34, their overall conformations are distinct from those of T. acidophilum Pelota and S. cerevisiae Dom34. Different overall conformations are due to conformational flexibility of the two linker regions between domains 1 and 2 and between domains 2 and 3. The observed inter-domain structural plasticity of Pelota proteins suggests that large conformational changes are essential for their functions.     

Immunogenic properties of archaeal species found in bioaerosols

The etiology of bioaerosol-related pulmonary diseases remains poorly understood. Recently, archaea emerged as prominent airborne components of agricultural environments, but the consequences of airway exposure to archaea remain unknown. Since subcomponents of archaea can be immunogenic, we used a murine model to study the pulmonary immune responses to two archaeal species found in agricultural facilities: Methanobrevibacter smithii (MBS) and Methanosphaera stadtmanae (MSS). Mice were administered intranasally with 6.25, 25 or 100 μg of MBS or MSS, once daily, 3 days a week, for 3 weeks. MSS induced more severe histopathological alterations than MBS with perivascular accumulation of granulocytes, pronounced thickening of the alveolar septa, alveolar macrophages accumulation and increased perivascular mononucleated cell accumulation. Analyses of bronchoalveolar lavage fluids revealed up to 3 times greater leukocyte accumulation with MSS compared to MBS. Instillation of 100 μg of MBS or MSS caused predominant accumulation of monocyte/macrophages (4.5×10(5) and 4.8×10(5) cells/ml respectively) followed by CD4(+) T cells (1.38×10(5) and 1.94×10(5) cells/ml respectively), B cells (0.73×10(5) and 1.28×10(5) cells/ml respectively), and CD8(+) T cells (0.20×10(5) and 0.31×10(5) cells/ml respectively) in the airways. Both archaeal species induced similar titers of antigen-specific IgGs in plasma. MSS but not MBS caused an accumulation of eosinophils and neutrophils in the lungs, which surprisingly, correlated inversely with the size of the inoculum. Stronger immunogenicity of MSS was confirmed by a 3 fold higher accumulation of myeloid dendritic cells in the airways, compared to MBS. Thus, the dose and species of archaea determine the magnitude and nature of the pulmonary immune response. This is the first report of an immunomodulatory role of archaeal species found in bioaerosols.     

Comparison of the structural basis for thermal stability between archaeal and bacterial proteins

In this study, the structural basis for thermal stability in archaeal and bacterial proteins was investigated. There were many common factors that confer resistance to high temperature in both archaeal and bacterial proteins. These factors include increases in the Lys content, the bends and blanks of secondary structure, the Glu content of salt bridge; decreases in the number of main–side chain hydrogen bond and exposed surface area, and changes in the bends and blanks of amino acids. Certainly, the utilization of charged amino acids to form salt bridges is a primary factor. In both heat-resistant archaeal and bacterial proteins, most Glu and Asp participate in the formation of salt bridges. Other factors may influence either archaeal or bacterial protein thermostability, which includes the more frequent occurrence of shorter 3₁₀-helices and increased hydrophobicity in heat-resistant archaeal proteins. However, there were increases in average helix length, the Glu content in salt bridges, temperature factors and decreases in the number of main–side chain hydrogen bonds, uncharged–uncharged hydrogen bonds, hydrophobicity, and buried and exposed polar surface area in heat-resistant bacterial proteins. Evidently, there are few similarities and many disparities between the heat-resistant mechanisms of archaeal and bacterial proteins.     

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division

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Highlights? A conserved protein, CdvA, recruits ESCRT-III to membranes during cell division ? Recruitment is mediated by a peptide-winged helix domain interaction ? We have determined the structure of this complex ? CdvA and a single ESCRT-III protein can drive membrane deformation in vitro     

Mechanical and failure properties of extracellular matrix sheets as a function of structural protein composition

The goal of this study was to determine how alterations in protein composition of the extracellular matrix (ECM) affect its functional properties. To achieve this, we investigated the changes in the mechanical and failure properties of ECM sheets generated by neonatal rat aortic smooth muscle cells engineered to contain varying amounts of collagen and elastin. Samples underwent static and dynamic mechanical measurements before, during, and after 30 min of elastase digestion followed by a failure test. Microscopic imaging was used to measure thickness at two strain levels to estimate the true stress and moduli in the ECM sheets. We found that adding collagen to the ECM increased the stiffness. However, further increasing collagen content altered matrix organization with a subsequent decrease in the failure strain. We also introduced collagen-related percolation in a nonlinear elastic network model to interpret these results. Additionally, linear elastic moduli correlated with failure stress which may allow the in vivo estimation of the stress tolerance of ECM. We conclude that, in engineered replacement tissues, there is a tradeoff between improved mechanical properties and decreased extensibility, which can impact their effectiveness and how well they match the mechanical properties of native tissue.     

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.     

The oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs)

Intron splicing is a prime example of the many types of RNA processing catalyzed by small nuclear ribonucleoprotein (snRNP) complexes. Sm proteins form the cores of most snRNPs, and thus to learn principles of snRNP assembly we characterized the oligomerization and ligand-binding properties of Sm-like archaeal proteins (SmAPs) from Pyrobaculum aerophilum (Pae) and Methanobacterium thermautotrophicum (Mth). Ultracentrifugation shows that Mth SmAP1 is exclusively heptameric in solution, whereas Pae SmAP1 forms either disulfide-bonded 14-mers or sub-heptameric states (depending on the redox potential). By electron microscopy, we show that Pae and Mth SmAP1 polymerize into bundles of well ordered fibers that probably form by head-to-tail stacking of heptamers. The crystallographic results reported here corroborate these findings by showing heptamers and 14-mers of both Mth and Pae SmAP1 in four new crystal forms. The 1.9 A-resolution structure of Mth SmAP1 bound to uridine-5'-monophosphate (UMP) reveals conserved ligand-binding sites. The likely RNA binding site in Mth agrees with that determined for Archaeoglobus fulgidus (Afu) SmAP1. Finally, we found that both Pae and Mth SmAP1 gel-shift negatively supercoiled DNA. These results distinguish SmAPs from eukaryotic Sm proteins and suggest that SmAPs have a generic single-stranded nucleic acid-binding activity.     

Engineering the allosteric properties of archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases

The archaeal non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN, EC 1.2.1.9) is a highly allosteric enzyme activated by glucose 1-phosphate (Glc1P). Recent kinetic analyses of two GAPN homologs from Sulfolobales show different allosteric behaviors toward the substrate glyceraldehyde-3-phosphate (GAP) and the allosteric effector Glc1P. In GAPN from Sulfolobus tokodaii (Sto-GAPN), Glc1P-induced activation follows an increase in affinity for GAP rather than an increase in maximum velocity, whereas in GAPN from Sulfolobus solfataricus (Sso-GAPN), Glc1P-induced activation follows an increase in maximum velocity rather than in affinity for GAP. To explore the molecular basis of this difference between Sto-GAPN and Sso-GAPN, we generated 14 mutants and 2 chimeras. The analyses of chimeric GAPNs generated from regions of Sto-GAPN and Sso-GAPN indicated that a 57-residue module located in the subunit interface was clearly involved in their allosteric behavior. Among the point mutations in this modular region, the Y139R variant of Sto-GAPN no longer displayed a sigmoidal K-type-like allostery, but instead had apparent V-type allostery similar to that of Sso-GAPN, suggesting that the residue located in the center of the homotetramer critically contributes to the allosteric behavior.     

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 A upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.     

Mechanical properties of actin

We used a cone and plate rheometer to evaluate the mechanical properties of actin over a wide range of oscillation frequencies and shear rates. Remarkably, both filamentous and nonfilamentous actin behaved as viscoelastic solids in both oscillatory and shear type experiments, providing that they were given ample time to equilibrate. Actin was purified by gel filtration from rabbit skeletal muscle and Acanthamoeba. Nonfilamentous actin in 2 different buffers had similar properties. In a low ionic strength buffer the absence of filaments was confirmed by electron microscopy, ultracentrifugation, and the fluorescence of pyrene-labeled actin. In 0.6 M KI, actin was monomeric by gel filtration. Filamentous actin had similar properties in 2 mM MgCl2 with either 50 mM KC1 or 500 mM KC1. Under all 4 of these conditions, actin required about 1000 min at 25 degrees C for the rheological properties to equilibrate. Under conditions where the oscillation of the rheometer did not affect the mechanical properties, all of the actin preparations had dynamic viscosities that were inverse functions of the frequency and dynamic elasticites that leveled off at low frequencies as expected for viscoelastic solids. For filamentous actin, the values of these parameters were about 2 times higher than for nonfilamentous actin. In shear experiments, both filamentous and nonfilamentous actin exhibited shear rate-dependent yield stresses. When filamentous and nonfilamentous actin structures were disrupted by transient shearing, the dynamic elasticity recovered to 90% in 30 min. Ovalbumin in the low ionic strength buffer also behaved as a viscoelastic material with elasticity and viscosity about 10 times lower than nonfilamentous actin, while cytochrome c behaved as a Newtonian fluid with a viscosity of 0.02 poise.     

Characterization of RNA-binding properties of the archaeal Hfq-like protein from Methanococcus jannaschii

The Sm and Sm-like proteins are widely distributed among bacteria, archaea and eukarya. They participate in many processes related to RNA-processing and regulation of gene expression. While the function of the bacterial Lsm protein Hfq and eukaryotic Sm/Lsm proteins is rather well studied, the role of Lsm proteins in Archaea is investigated poorly. In this work, the RNA-binding ability of an archaeal Hfq-like protein from Methanococcus jannaschii has been studied by X-ray crystallography, anisotropy fluorescence and surface plasmon resonance. It has been found that MjaHfq preserves the proximal RNA-binding site that usually recognizes uridine-rich sequences. Distal adenine-binding and lateral RNA-binding sites show considerable structural changes as compared to bacterial Hfq. MjaHfq did not bind mononucleotides at these sites and would not recognize single-stranded RNA as its bacterial homologues. Nevertheless, MjaHfq possesses affinity to poly(A) RNA that seems to bind at the unstructured positive-charged N-terminal tail of the protein.     

Morphology and structural properties of rafts

Working out of the role of lipids incorporated into membranes and the formation of a new view on the morphology, organization, and functioning of membranes has in the recent past taken place thanks to active study of the basic membrane clusters, rafts. In this overview, current data on the morphology and biophysical and biochemical features of rafts are summarized and the structure, form, and basic marker proteins of functional microdomains are described.     

The structural mechanism of the inhibition of archaeal RelE toxin by its cognate RelB antitoxin

The archaeal toxin, aRelE, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 inhibits protein synthesis, whereas its cognate antitoxin, aRelB, neutralizes aRelE activity by forming a non-toxic complex, aRelB-aRelE. The structural mechanism whereby aRelB neutralizes aRelE activity was examined by biochemical and biophysical analyses. Overexpression of aRelB with an aRelE mutant (ΔC6), in which the C-terminal residues critical for aRelE activity were deleted, in Escherichia coli allowed a stable complex, aRelB-ΔC6, to be purified. Isothermal titration of aRelE or ΔC6 with aRelB indicated that the association constant (Ka) of wild-type aRelB-aRelE is similar to that of aRelB-ΔC6, demonstrating that aRelB makes little contact with the C-terminal active site of aRelE. Overexpression of deletion mutants of aRelB with aRelE indicated that either the N-terminal (pos. 1-27) or C-terminal (pos. 50-67) fragment of aRelB is sufficient to counteract the toxicity of aRelE in E. coli cells and the second α-helix (α2) in aRelB plays a critical role in forming a stable complex with aRelE. The present results demonstrate that aRelB, as expected from its X-ray structure, precludes aRelE from entering the ribosome, wrapping around the molecular surface of aRelE.     

Molecular and functional properties of an archaeal phenylalanyl-tRNA synthetase from the hyperthermophile Sulfolobus solfataricus

An archaeal phenylalanyl-tRNA synthetase (FRS) has been purified from the hyperthermophile Sulfolobus solfataricus (Ss). This enzyme is a heterotetramer made of two different subunits whose molecular mass is 56 kDa and 64 kDa, respectively. As thought, SsFRS is essential for the in vitro poly(Phe) synthesis. Interestingly, the enzyme is able to aminoacylate only endogenous tRNA but it does not seem to be a strictly ATP-dependent synthetase. SsFRS interacts with the elongation factor 1alpha isolated from the same source; this caused a significant enhancement of the SstRNA aminoacylation efficiency, thus indicating that, as well as in eukarya, in this archaeon a tRNA channelling mechanism should occur. The overall results presented in this paper show that the archaeal SsFRS behaves as the analogous enzymes isolated from eukaryal sources rather than those from eubacterial organisms.     

Interactions between the archaeal Cdc6 and MCM proteins modulate their biochemical properties

The origin recognition complex, Cdc6 and the minichromosome maintenance (MCM) complex play essential roles in the initiation of eukaryotic DNA replication. Homologs of these proteins may play similar roles in archaeal replication initiation. While the interactions among the eukaryotic initiation proteins are well documented, the protein–protein interactions between the archaeal proteins have not yet been determined. Here, an extensive structural and functional analysis of the interactions between the Methanothermobacter thermautotrophicus MCM and the two Cdc6 proteins (Cdc6-1 and -2) identified in the organism is described. The main contact between Cdc6 and MCM occurs via the N-terminal portion of the MCM protein. It was found that Cdc6–MCM interaction, but not Cdc6–DNA binding, plays the predominant role in regulating MCM helicase activity. In addition, the data showed that the interactions with MCM modulate the autophosphorylation of Cdc6-1 and -2. The results also suggest that MCM and DNA may compete for Cdc6-1 protein binding. The implications of these observations for the initiation of archaeal DNA replication are discussed.