Modulation of Conformational Equilibria in the S-Adenosylmethionine (SAM) II Riboswitch by SAM, Mg(2+), and Trimethylamine N-Oxide.

The dependence of the conformation of the S-adenosylmethionine (SAM) II riboswitch on the concentration of added Mg(2+) ions and SAM, individually and in mixtures, was monitored by circular dichroism (CD) spectroscopy and by measurement of the diffusion coefficient. The results are analyzed in the context of two complementary quantitative models, both of which are consistent with a single underlying physical model. Magnesium binding sites in the open state have an affinity on average higher than the affinity of those in the compact state, but formation of the compact state is accompanied by an increase in the number of binding sites.

The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology.

Simplagrin, a platelet aggregation inhibitor from Simulium nigrimanum salivary glands specifically binds to the Von Willebrand factor receptor in collagen and inhibits carotid thrombus formation in vivo.

Background: Among the several challenges faced by bloodsucking arthropods, the vertebrate hemostatic response against blood loss represents an important barrier to efficient blood feeding. Here we report the first inhibitor of collagen-induced platelet aggregation derived from the salivary glands of a black fly (Simulium nigrimanum), named Simplagrin.

Methods And Findings: Simplagrin was expressed in mammalian cells and purified by affinity-and size-exclusion chromatography.

Single-step total fractionation of single-wall carbon nanotubes by countercurrent chromatography.

Development of simple processes to fractionate synthetic mixtures of single-wall carbon nanotubes (SWCNTs) into individual species is crucial to many applications. Existing methods for single-chirality SWCNT purification are cumbersome, often requiring multiple steps and different conditions for different species. Here, we report a method to achieve total fractionation of a synthetic SWCNT mixture by countercurrent chromatography, resulting in purification of many single-chirality SWCNT species in a single run.

Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex.

Saccharomyces cerevisiae antizyme (AZ) resembles mammalian AZ in its mode of synthesis by translational frameshifting and its ability to inhibit and facilitate the degradation of ornithine decarboxylase (ODC). Despite many studies on the interaction of AZ and ODC, the ODC:AZ complex has not been purified from any source and thus clear information about the stoichiometry of the complex is still lacking. In this study we have studied the yeast antizyme protein and the ODC:AZ complex.

Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH.

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro.

Effects of hydrostatic pressure on the conformational equilibrium of tryptophan synthase from Salmonella typhimurium.

A wide range of parameters influence allosteric communications between the alpha- and beta-subunits of the Trp synthase alpha(2)beta(2) multienzyme complex with L-Ser, including monovalent cations, pH, temperature, ligands, organic solvents, and hydrostatic pressure. The conformational change from closed to open can be monitored either by absorbance at 423 nm or fluorescence at 495 nm from the pyridoxal-5'-phosphate-L-Ser complex. Pressure perturbation was used to quantify the effects of monovalent cations, ligands, and mutations on the conformational equilibrium of Trp synthase.

Aegyptin displays high-affinity for the von Willebrand factor binding site (RGQOGVMGF) in collagen and inhibits carotid thrombus formation in vivo.

Aegyptin is a 30 kDa mosquito salivary gland protein that binds to collagen and inhibits platelet aggregation. We have studied the biophysical properties of aegyptin and its mechanism of action. Light-scattering plot showed that aegyptin has an elongated monomeric form, which explains the apparent molecular mass of 110 kDa estimated by gel-filtration chromatography.

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis.

Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.

The functional curli amyloid is not based on in-register parallel beta-sheet structure.

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid.

Pressure and temperature jump relaxation kinetics of the conformational change in Salmonella typhimurium tryptophan synthase L-serine complex: large activation compressibility and heat capacity changes demonstrate the contribution of solvation.

Tryptophan synthase is an alpha2beta2 multienzyme complex that exhibits coupling of the alpha- and beta-subunit reactions by tightly controlled allosteric interactions. A wide range of parameters can affect the allosteric interactions, including monovalent cations, pH, alpha-site and beta-site ligands, temperature, and pressure. Rapid changes in hydrostatic pressure (P-jump) and temperature (T-jump) were used to examine the effects of pressure and temperature on the rates of the interconversion of external aldimine and aminoacrylate intermediates in the Tryptophan synthase-L-Ser complex.

Assembly of the yeast cell wall. Crh1p and Crh2p act as transglycosylases in vivo and in vitro.

The cross-linking of polysaccharides to assemble new cell wall in fungi requires mechanisms by which a preexisting linkage is broken for each new one made, to allow for the absence of free energy sources outside the plasma membrane. Previous work showed that Crh1p and Crh2p, putative transglycosylases, are required for the linkage of chitin to beta(1-3)glucose branches of beta(1-6)glucan in the cell wall of budding yeast. To explore the linking reaction in vivo and in vitro, we used fluorescent sulforhodamine-linked laminari-oligosaccharides as artificial chitin acceptors.

Concentration-independent estimation of protein secondary structure by circular dichroism: a comparison of methods.

Estimation of a protein's secondary structure from its circular dichroism spectrum usually requires accurate knowledge of the concentration and pathlength of the sample. Two recently described methods avoid this problem by analysis of g-factor spectra (McPhie, Anal. Biochem.

Nucleotidylylation of the VPg protein of a human norovirus by its proteinase-polymerase precursor protein.

Caliciviruses have a positive strand RNA genome covalently-linked at the 5'-end to a small protein, VPg. This study examined the biochemical modification of VPg by the ProPol form of the polymerase of human norovirus strain MD145 (GII.4).

Quantitative effects of allosteric ligands and mutations on conformational equilibria in Salmonella typhimurium tryptophan synthase.

Allosteric communications are important in coordination of the reactions in the tryptophan (Trp) synthase alpha2beta2 multienzyme complex. We have measured the conformational equilibria of l-Ser and l-Trp complexes, using absorption and fluorescence spectrophotometry with hydrostatic pressure equilibrium perturbation. The effects of monovalent cations, disodium alpha-glycerophosphate (Na2GP), indoleacetylglycine (IAG), and benzimidazole (BZI), as well as of betaE109D and betaD305A mutations, on K(eq) for the conformational equilibria were determined.

Control of in vitro immune responses by regulatory oligodeoxynucleotides through inhibition of pIII promoter directed expression of MHC class II transactivator in human primary monocytes.

Ag presentation is a key step in the initiation of adaptive immune responses that depends on the expression of MHC Ags and costimulatory molecules. Immune-enhancing CpG and non-CPG oligodeoxynucleotides (ODNs) stimulate Ag presentation by stimulating the expression of these molecules and by promoting dendritic cell maturation. In this report, we identify immunoregulatory orthophosphorothioate non-CpG molecules, referred to as regulatory ODNs (rODNs), by their ability to inhibit allogeneic monocyte-stimulated T cell responses and down-regulate HLA-DR in human primary monocytes.

Macromolecular crowding stabilizes the molten globule form of apomyoglobin with respect to both cold and heat unfolding.

At pH 2 apomyoglobin is extensively unfolded. Addition of increasing concentration of salts has been shown to convert the protein into molten globule form(s), which can undergo both heat-induced and cold-induced unfolding. Increasing concentrations of an inert polymer, dextran, lead to increased formation of molten globule and stabilizes the protein with respect to both heat-induced and cold-induced denaturation.

Deoxyhypusine hydroxylase is a Fe(II)-dependent, HEAT-repeat enzyme. Identification of amino acid residues critical for Fe(II) binding and catalysis [corrected].

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (N(epsilon)-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs.

The AUUCU repeats responsible for spinocerebellar ataxia type 10 form unusual RNA hairpins.

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat.

Thiol-disulphide interchange in tubulin: kinetics and the effect on polymerization.

All 20 cysteine residues are accessible to disulphide reagents in the tubulin dimer, whereas only four are accessible in taxol-stabilized microtubules. Reaction rates with disulphide reagents are a function of the reagent, are decreased by G nucleotides, and increased with increase in pH and urea. With transient (stop-flow) kinetics, DTNB [5,5'-dithiobis-(2-nitrobenzoic acid)] and 2,2'-dithiodipyridine progress curves cannot be fitted by the sum of exponential terms based only on classes of cysteines.

Stable secondary structure near the nicking site for adeno-associated virus type 2 Rep proteins on human chromosome 19.

Adeno-associated virus serotype 2 (AAV-2) can preferentially integrate its DNA into a 4-kb region of human chromosome 19, designated AAVS1. The nicking activity of AAV-2's Rep68 or Rep78 proteins is essential for preferential integration. These proteins nick at the viral origin of DNA replication and at a similar site within AAVS1.

CD studies on films of amyloid proteins and polypeptides: quantitative g-factor analysis indicates a common folding motif.

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a "cross-beta" structure, with extended beta-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P.

Multi-tissue gene-expression analysis in a mouse model of thyroid hormone resistance.

Background: Resistance to thyroid hormone (RTH) is caused by mutations of the thyroid hormone receptor beta (TRbeta) gene. To understand the transcriptional program underlying TRbeta mutant-induced phenotypic expression of RTH, cDNA microarrays were used to profile the expression of 11,500 genes in a mouse model of human RTH.

Results: We analyzed transcript levels in cerebellum, heart and white adipose tissue from a knock-in mouse (TRbetaPV/PV mouse) that harbors a human mutation (referred to as PV) and faithfully reproduces human RTH.

Co-assembly of envoplakin and periplakin into oligomers and Ca(2+)-dependent vesicle binding: implications for cornified cell envelope formation in stratified squamous epithelia.

Plakin family members envoplakin and periplakin have been shown to be part of the cornified cell envelope in terminally differentiating stratified squamous epithelia. In the present study, purified recombinant human envoplakin and periplakin were used to investigate their properties and interactions. We found that envoplakin was insoluble at physiological conditions in vitro, and co-assembly with periplakin was required for its solubility.