16 research outputs found

    A designed phenylalanyl-tRNA synthetase variant allows efficient in vivo incorporation of aryl ketone functionality into proteins

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    Incorporation of non-natural amino acids into proteins in vivo expands the scope of protein synthesis and design. p-Acetylphenylalanine was incorporated into recombinant dihydrofolate reductase (DHFR) in Escherichia coli via a computationally designed mutant form of the phenylalanyl-tRNA synthetase of the host. DHFR outfitted with ketone functionality can be chemoselectively ligated with hydrazide reagents under mild conditions

    Derivatives of 8-Hydroxy-2-methylquinoline Are Powerful Prototypes for Zinc Sensors in Biological Systems

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    The recent emphasis on understanding the myriad roles of zinc in both normal and diseased cells and tissues has placed an ever increasing demand on methods for sensitive and selective methods for real-time monitoring of free Zn^(2+) in complex biological samples. Chelation-enhanced fluorescent sensors for zinc, based on fluorophores such as quinoline, dansyl, fluorescein, and anthracene, have been reported. While each of these agents has unique advantages, there remain issues with sensitivity, selectivity, and specificity that may be addressable with an alternate chromophore that is readily amenable to synthetic manipulation. Herein we report the systematic chemical modification of the 8-hydroxy-2-methylquinoline (Oxn) unit as a building block for the development of new sensors employing chelation-enhanced fluorescence. In particular, improvements in quantum yield from 0.004 to 0.70 and stepwise blue shifts in fluorescence emission wavelengths (to a total of over 70 nm) are reported

    Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

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    Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Forster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions

    Lithographic Patterning of Photoreactive Cell-Adhesive Proteins

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    We describe a novel, simple method for the photolithographic patterning of cell-adhesive proteins. Intrinsically photoreactive proteins are synthesized in Escherichia coli through incorporation of the non-canonical, photosensitive amino acid para-azidophenylalanine. Upon ultraviolet irradiation at 365 nm, proteins form cross-linked films with elastic moduli that can be tuned by varying the concentration of photoreactive amino acid in the expression medium. Films of these proteins can be directly patterned using standard photolithographic techniques. We demonstrate the utility of this method of protein patterning by creating stable arrays of fibroblast cells on an engineered protein “photoresist”

    Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogues

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    Unnatural amino acids with useful chemical functionality can replace phenylalanine in bacterial proteins. Coexpression of a promiscuous phenylalanine-tRNA synthetase mutant enables the synthesis of target proteins bearing iodophenyl, cyanophenyl, ethynylphenyl, azidophenyl, and pyridyl groups (see general structures). Proteins incorporating the analogues have a range of potential applications, including Pd-mediated conjugation (R=CCH), photoaffinity labeling (R=N_3), X-ray phasing (R=I), and novel metal coordination (R=pyridyl)

    Light-Activated StaudingerBertozzi Ligation within Living Animals

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    Altering Adenoviral Tropism via Click Modification with ErbB Specific Ligands

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    Methods for targeting oncolytic viruses can increase efficacy and accelerate development. Genetic engineering, the predominant method for changing vector tropism, is limited in scope and often represents the bottleneck for vector development. Metabolic incorporation of an unnatural azido sugar, <i>O-</i>GlcNAz, at a specific site on the adenoviral surface allows chemoselective attachment of affibodies for Her2 or EGF receptors. Modification with these high-affinity, high-selectivity proteins is straightforward and readily generalizable, demonstrates minimal impact on virus physiology, and affords significant increases in gene delivery to cancer cells. As a result, this method has significant potential to increase the efficacy of next-generation viral vectors

    Light-Activated Staudinger–Bertozzi Ligation within Living Animals

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    The ability to regulate small molecule chemistry <i>in vivo</i> will enable new avenues of exploration in imaging and pharmacology. However, realization of these goals will require reactions with high specificity and precise control. Here we demonstrate photocontrol over the highly specific Staudinger–Bertozzi ligation <i>in vitro</i> and <i>in vivo</i>. Our simple approach, photocaging the key phosphine atom, allows for the facile production of reagents with photochemistry that can be engineered for specific applications. The resulting compounds, which are both stable and efficiently activated, enable the spatial labeling of metabolically introduced azides <i>in vitro</i> and on live zebrafish
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