Immobilizing reporters for molecular imaging of the extracellular microenvironment in living animals.

We report here an immobilization strategy using a collagen binding protein to deliver and confine synthetic reporters to the extracellular microenvironment in vivo for noninvasively imaging the activity of targets in the microenvironment. We show that the immobilization of reporters on collagens in the local microenvironment is highly efficient and physiologically stable for repetitive, long-term imaging. By using this strategy we successfully developed an immobilized bioluminescent activatable reporter and a dual-modality reporter to map and quantitatively image the activity of extracellular matrix metalloproteinases (MMP) in tumor-bearing mice.

Bioluminescent nanosensors for protease detection based upon gold nanoparticle-luciferase conjugates.

This communication reports the use of click chemistry to site-specifically conjugate bioluminescent Renilla luciferase proteins to gold nanoparticles (Au NPs) for sensing protease activity. The bioluminescent emission from luciferase was efficiently quenched by Au NPs, but significantly recovered after the proteolytic cleavage.

A biocompatible condensation reaction for the labeling of terminal cysteine residues on proteins.

This communication describes a novel protein labeling method that uses a single amino-acid tag — N-terminal cysteine residue — and small-molecule probes carrying the cyanobenzothiazole unit for specific labeling of proteins in vitro and at the surface of live cells. This simple ligation reaction proceeds with a high degree of specificity in physiological conditions, and should offer an important alternative to currently available protein labeling methods.

Semiconductor quantum dots for biosensing and in vivo imaging.

Semiconductor quantum dots (QDs) have captivated researchers in the biomedical field over the last decade. Compared to organic dyes and fluorescent proteins, QDs have unique optical properties such as tunable emission spectra, improved brightness, superior photostability, and simultaneous excitation of multiple fluorescence colors. Since the first successful reports on the biological use of QDs a decade ago, QDs and their bioconjugates have been successfully applied to various imaging applications including fixed cell labeling, live-cell imaging, in situ tissue profiling, fluorescence detection and sensing, and in vivo animal imaging.

Biosensing and imaging based on bioluminescence resonance energy transfer.

Bioluminescence resonance energy transfer (BRET) operates with biochemical energy generated by bioluminescent proteins to excite fluorophores and offers additional advantages over fluorescence energy transfer (FRET) for in vivo imaging and biosensing. While fluorescent proteins are frequently used as BRET acceptors, both small molecule dyes and nanoparticles can also serve as acceptor fluorophores. Semiconductor fluorescent nanocrystals or quantum dots (QDs) are particularly well suited for use as BRET acceptors due to their high quantum yields, large Stokes shifts and long wavelength emission.

Multiplex detection of protease activity with quantum dot nanosensors prepared by intein-mediated specific bioconjugation.

We report here a protease sensing nanoplatform based on semiconductor nanocrystals or quantum dots (QDs) and bioluminescence resonance energy transfer (QD-BRET) to detect the protease activity in complex biological samples. These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and protease substrates sandwiched between the two as a sensing group. An intein-mediated conjugation strategy was developed for site-specific conjugation of proteins to QDs in preparing these QD nanosensors.

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction.

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V.

Mutational, structural, and kinetic evidence for a dissociative mechanism in the GDP-mannose mannosyl hydrolase reaction.

GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-alpha-d-sugars by nucleophilic substitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124. Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.

Structure and mechanism of GDP-mannose glycosyl hydrolase, a Nudix enzyme that cleaves at carbon instead of phosphorus.

GDP-mannose glycosyl hydrolase (GDPMH) catalyzes the hydrolysis of GDP-mannose and GDP-glucose to GDP and sugar by substitution with inversion at C1 of the sugar. The enzyme has a modified Nudix motif and requires one divalent cation for activity. The 1.

Lipoxygenase-catalyzed polymerization of phenolic lipids suggests a new mechanism for allergic contact dermatitis induced by urushiol and its analogs.

Lipoxygenase was found to catalyze the oxidative polymerization of phenolic lipids containing a (Z,Z)-pentadiene in the side chain, the model compounds of urushiol and its analog, yielding methanol-soluble and insoluble polymers. The structural analysis of the resulted polymers suggested that the polymerization occurred at both the phenol and the unsaturated side chain. The key step of the polymerization was the generation of the hydroperoxide at the unsaturated side chain by lipoxygenase.

Enzymatic synthesis of polyphenols from highly phenolic lignin-based polymers (lignophenols).

Peroxidase-catalyzed polymerization of lignin-based macromonomers (lignophenols), lignocatechol and lignocresol, prepared by phenolation of lignin with catechol or p-cresol, was carried out in aqueous organic solvent mixtures. The two lignophenols were polymerized to give cross-linked polymers. The highest yield of polymerization (83%, w/w) was obtained with lignocatechol, and the maximum yield for the polymerization of lignocresol was 55% (w/w).