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Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.
ACS Chem Biol 2008 Sep 19
PMID:Chain dynamics of nascent polypeptides emerging from the ribosome. 1880 69

Although the vital roles of structures containing sialic acid in biomolecular recognition are well documented, limited information is available on how sialic acid structural modifications, sialyl linkages, and the underlying glycan structures affect the binding or the activity of sialic acid-recognizing proteins and related downstream biological processes. A novel combinatorial chemoenzymatic method has been developed for the highly efficient synthesis of biotinylated sialosides containing different sialic acid structures and different underlying glycans in 96-well plates from biotinylated sialyltransferase acceptors and sialic acid precursors. By transferring the reaction mixtures to NeutrAvidin-coated plates and assaying for the yields of enzymatic reactions using lectins recognizing sialyltransferase acceptors but not the sialylated products, the biotinylated sialoside products can be directly used, without purification, for high-throughput screening to quickly identify the ligand specificity of sialic acid-binding proteins. For a proof-of-principle experiment, 72 biotinylated alpha2,6-linked sialosides were synthesized in 96-well plates from 4 biotinylated sialyltransferase acceptors and 18 sialic acid precursors using a one-pot three-enzyme system. High-throughput screening assays performed in NeutrAvidin-coated microtiter plates show that whereas Sambucus nigra Lectin binds to alpha2,6-linked sialosides with high promiscuity, human Siglec-2 (CD22) is highly selective for a number of sialic acid structures and the underlying glycans in its sialoside ligands.
ACS Chem Biol 2008 Sep 19
PMID:Combinatorial chemoenzymatic synthesis and high-throughput screening of sialosides. 1872 52

The ability to incorporate chemical probes into peptides is of great importance because it can render novel functionality to proteins and greatly expand our capacity to investigate complex biological systems. A methodology developed by the Schultz laboratory provides a unique strategy to incorporate chemical probes as unnatural amino acids into proteins by "expanding the genetic code" of the host cell. A recent application of this methodology that allows the site-specific incorporation of three NMR-active probes into proteins demonstrates the potential for researchers to explore avenues that are not easily achievable with existing methods.
ACS Chem Biol 2008 Sep 19
PMID:Site-specific incorporation of chemical probes into proteins for NMR. 1880 68

Deciphering the mechanism of folding of newly synthesized proteins in the cell is a major challenge because of the large size and multiplicity of molecular components involved and the asynchrony of biosynthesis. Fluorescently labeled ribosome-bound nascent chains of a defined length were prepared and subjected to dynamic fluorescence depolarization spectroscopy measurements. Nanosecond anisotropy decay correlation times of proteins' nascent chains at different stages of polypeptide elongation were determined for the first time. Striking dependence of the chain dynamics on the stages of elongation was observed and revealed chain length dependence of folding on the ribosome.
ACS Chem Biol 2008 Sep 19
PMID:Folding on the assembly line. 1871 65

The precise mechanisms by which omega-3 fatty acids improve fat metabolism are not completely understood. This study was designed to determine the effects of eicosapentaenoic acid (EPA) ethyl ester administration on the expression levels of several muscle, liver and adipose tissue genes involved in lipogenesis and fatty acid oxidation pathways. Male Wistar rats fed a standard diet (control animals) or a high-fat diet were treated daily by oral gavage with EPA ethyl ester (1g/kg) for 5 weeks. The high-fat diet caused a very significant increase in plasma cholesterol (P<.01) levels, which was reverted by EPA (P<.001). A significant decrease in circulating triglyceride levels (P<.05) was also observed in EPA-treated groups. EPA administration induced a significant down-regulation in some lipogenic genes such as muscle acetyl CoA carboxylase beta (ACC beta) (P<.05) and liver fatty acid synthase (FAS) (P<.05). Furthermore, a decrease in glucokinase (GK) gene expression was observed in EPA-treated animals fed a control diet (P<.01), whereas a significant increase in GK mRNA levels was found in groups fed a high-fat diet. On the other hand, no alterations in genes involved in beta-oxidation, such acetyl CoA synthase 4 (ACS4), acetyl CoA synthase 5 (ACS5) or acetyl CoA oxidase (ACO), were found in EPA-treated groups. Surprisingly and opposite to the expectations, a very significant decrease in the expression levels of liver PPARalpha (P<.01) was observed after EPA treatment. These findings show the ability of EPA ethyl ester treatment to down-regulate some genes involved in fatty acid synthesis without affecting the transcriptional activation of beta-oxidation-related genes.
J Nutr Biochem 2009 Sep
PMID:Down-regulation in muscle and liver lipogenic genes: EPA ethyl ester treatment in lean and overweight (high-fat-fed) rats. 1882 85

Real-time study of the transport and biocompatibility of nanomaterials in early embryonic development at single-nanoparticle resolution can offer new knowledge about the delivery and effects of nanomaterials in vivo and provide new insights into molecular transport mechanisms in developing embryos. In this study, we directly characterized the transport of single silver nanoparticles into an in vivo model system (zebrafish embryos) and investigated their effects on early embryonic development at single-nanoparticle resolution in real time. We designed highly purified and stable (not aggregated and no photodecomposition) nanoparticles and developed single-nanoparticle optics and in vivo assays to enable the study. We found that single Ag nanoparticles (5-46 nm) are transported into and out of embryos through chorion pore canals (CPCs) and exhibit Brownian diffusion (not active transport), with the diffusion coefficient inside the chorionic space (3 x 10(-9) cm(2)/s) approximately 26 times lower than that in egg water (7.7 x 10(-8) cm(2)/s). In contrast, nanoparticles were trapped inside CPCs and the inner mass of the embryos, showing restricted diffusion. Individual Ag nanoparticles were observed inside embryos at each developmental stage and in normally developed, deformed, and dead zebrafish, showing that the biocompatibility and toxicity of Ag nanoparticles and types of abnormalities observed in zebrafish are highly dependent on the dose of Ag nanoparticles, with a critical concentration of 0.19 nM. Rates of passive diffusion and accumulation of nanoparticles in embryos are likely responsible for the dose-dependent abnormalities. Unlike other chemicals, single nanoparticles can be directly imaged inside developing embryos at nanometer spatial resolution, offering new opportunities to unravel the related pathways that lead to the abnormalities.
ACS Nano 2007 Sep
PMID:In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. 1912 72

Single-particle tracking of individual single-walled carbon nanotubes (SWNTs) using their near-infrared band gap fluorescence is a powerful tool for understanding how these Brownian rods diffuse and interact with various molecular force potentials, including living systems. Pioneered by the Weisman laboratory at Rice University, the method is one of the only available to study single SWNT molecules in solution over extended periods since SWNTs have no apparent irreversible photobleaching threshold at moderate fluence and no intrinsic blinking mechanism. Recent progress by Tsyboulski et al. shows how real-time measurement of rotational and transitional diffusivities can provide information about rod length and mechanical properties. Recently, Jin et al. used single-particle tracking to map the trajectories of SWNTs as they are incorporated into and expelled from NIH-3T3 cells in real time. The technique has provided the first evidence of nanoparticle exocytosis in this case and demonstrates an expulsion rate that closely matches the endocytosis rate. The ability to track and to analyze single molecules in this way may lead to new technologies that utilize as their platform a single, freely diffusing nanotube.
ACS Nano 2008 Sep 23
PMID:Where is it heading? Single-particle tracking of single-walled carbon nanotubes. 1920 15

Recent advances in probing properties at very high spatial resolution are enabling remarkable progress in understanding local physical and chemical phenomena. Additionally, these observations raise questions as to the ultimate limit of resolution in what are considered continuum properties. As complex property probes achieve increasingly high spatial resolution, they approach the transition between continuum and atomistic descriptions of properties. The recent observations imply that further advances are imminent.
ACS Nano 2008 Sep 23
PMID:Pushing resolution limits of functional imaging to probe atomic scale properties. 1920 13

A straightforward and effective polyol route for the controllable synthesis of high-quality gold (Au) octahedra with uniform size is presented in an ethylene glycol solution. Large-scale Au octahedra with the size ranging from tens to hundreds of nanometers were selectively synthesized in high-yield. The surfaces of octahedral Au nanocrystals are smooth and correspond to {111} planes. Formation of Au nanooctahedra was attributed to the preferential adsorption of cationic surfactant poly(diallyldimethylammonium) chloride (PDDA) molecules on the {111} planes of Au nuclei that inhibited the growth rate along the <111> direction. The reduction rate of gold ions in the synthesis process can be rationally manipulated by acidic and basic solutions. This provides a facile and effective route to harvest Au octahedra with different dimensions. The synthetic strategy has the advantage of one-pot and requires no seeds, no foreign metal ions, and no pretreatment of the precursor, so that this is a practical method for controllable synthesis of Au octahedra. Size-dependent optical properties of Au octahedra were numerically and experimentally analyzed. The analysis shows that Au octahedra with sharp edges possess attractive optical properties, promising their applications to surface-enhancement spectroscopy, chemical or biological sensing, and the fabrication of nanodevices.
ACS Nano 2008 Sep 23
PMID:A facile polyol route to uniform gold octahedra with tailorable size and their optical properties. 1920 14

Near-infrared fluorescence videomicroscopy has been used to study simultaneously the translational and rotational diffusion of individual semiconducting single-walled carbon nanotubes (SWCNTs) in aqueous suspension. Analysis of translational trajectories revealed diffusion coefficient values from approximately 0.3 to 6 microm(2)/s. The nanotube lengths deduced from these values ranged between approximately 130 nm and 6 microm. From the minor bending motions observed in individual nanotubes several micrometers in length, we confirmed that the shorter SWCNTs of primary interest here can be considered to be rigid rods under normal conditions. Because the nanotubes act as highly rigid, photostable, steady, and anisotropic fluorophores, it was possible to monitor their rotational reorientations through fluctuations in emission intensity under linearly polarized excitation. The magnitudes of observed orientational fluctuations varied substantially among individual nanotubes. These magnitudes correlated strongly with translational diffusion coefficient, reflecting the length dependence of both types of motions. Combined translational and rotational measurements also revealed the influence of local environment on nanotube mobility.
ACS Nano 2008 Sep 23
PMID:Translational and rotational dynamics of individual single-walled carbon nanotubes in aqueous suspension. 1920 12


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