Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the heme oxygenase responsible for BV biosynthesys and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared to monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labeling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumors in whole mice. Our work shows promise in the application of IFPs for protein labeling and in vivo imaging.

Rapid, sensitive, and sequence-specific DNA detection can be achieved in one step using an engineered intrasterically regulated enzyme. The semi-synthetic inhibitor-DNA-enzyme (IDE) construct (left) rests in the inactive state, but upon exposure to a complementary DNA sequence undergoes a DNA hybridization-triggered allosteric enzyme activation (right). The ensuing rapid substrate turnover provides the built-in signal amplification mechanism for detecting approximately 10 femtomoles of DNA in less than three minutes under physiological conditions. We describe the design, synthesis, and functional characterization of an intrasterically regulated semi-synthetic enzyme and its application in sequence specific DNA detection. 1-2 The system is composed of covalently associated inhibitor-DNA-enzyme (IDE) modules and functions via DNA hybridization-triggered allosteric enzyme activation and signal amplification through substrate turnover. 3 Its functional capacity is highlighted by the sequence specific detection of approximately 10 femtomoles of DNA in less than three minutes under physiological conditions. These studies suggest that rationally designed intrasterically regulated enzymes may constitute a promising new class of reagents for highly sensitive, rapid, and PCR-independent one-step detection of label-free DNA sequences.The principles that govern intrasteric regulation in natural enzymes 4 are firmly rooted in the physicochemical advantages of intramolecular binding. These enzymes often use an appending N-or C-terminal polypeptide pseudosubstrate to block access to their active sites. Enzyme regulation (activation) occurs at an allosteric site, typically the site between the enzyme and pseudosubstrate, where a conformational change or cleavage event removes the pseudosubstrate from the active site. We sought to mimic the intrasteric regulatory features of natural enzymes by designing novel semi-synthetic constructs that can be activated in the presence of complementary DNA sequences. The IDE complex described employs a single stranded DNA probe to covalently tether Cereus neutral protease 5 (CNP) to its small molecule NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phosphoramidite inhibitor. 6 We anticipated that the conformational flexibility of the ss-DNA tether in an appropriately functionalized system would allow facile intramolecular binding of the inhibitor to the enzyme active site to furnish the inactive state of the IDE (Figure 1). Likewise, we postulated that IDE should rapidly convert to an active state in the presence of its complementary DNA sequence because formation of a high-affinity DNA duplex structure was expected to drastically alter the conformation of the tether and favor the liberation of the inhibitor from the enzyme active site. Once activated, the enzyme would act upon its fluorogenic substrate, present in situ, producing an optical output signal. Moreover, because the enzyme turns over many copies of the substrate, in principle each probe hybridiza...

Understanding the specificity of cell-surface carbohydrates interaction with antibodies and receptors is important for the development of new therapeutics and high-sensitivity diagnostics. This approach is, however, limited to the availability of natural and truncated sequences of the oligosaccharides and the sensitivity of the assay system. Reported here is the synthesis of the cancer antigen Globo H hexasaccharide, an epitope found on the cell surface of breast, prostate, and ovarian cancers, and its truncated sequences by using the programmable one-pot synthesis strategy. The saccharides were then arrayed covalently on glass slides with different density and used for the fluorencense-based binding analysis of two monoclonal antibodies against Globo H and the serum from breast cancer patients, to define the specificity of these antibodies. It was shown that the terminal tetrasaccharide binds the monoclonal antibodies equally well as does the hexasaccharide and the fucose residue is required for effective binding. The serum binds both the defucosylated pentasaccharide and the fucosylated hexasaccharide without a significant difference, perhaps because of the polyclonal nature of the serum or the presence of diverse immune responses to different sugar epitopes at various stages. This method requires very small amounts of materials and is more effective and sensitive than the traditional ELISA method, and thus provides another platform to monitor the immune response to carbohydrate epitopes at different stages during differentiation, metastasis, or treatment.programmable one-pot synthesis ͉ glycoarray ͉ glycan epitope ͉ Globo H-truncated sequences T he cell-surface glycosphingolipid Globo H is a member of a family of antigenic carbohydrates that are highly expressed on a range of cancer cell lines, especially breast cancer cells (1-4). Furthermore, it has been established that the serum of breast cancer patients contains high levels of antibodies against the Globo H epitope, and this epitope is also targeted by the monoclonal antibodies MBr1 (5-7) and VK-9 (8). As a result, this hexasaccharide has been the focus of studies aimed at anticancer vaccine development (9-15). Many elegant syntheses of Globo H have been reported (11,(16)(17)(18)(19)(20)(21)(22)(23)(24), including one approach that uses the one-pot programmable oligosaccharide synthesis developed in our laboratory (25). Previously, it has been reported that certain truncated Globo H derivatives can still be effective in binding MBr1 and VK-9 antibodies, which could increase the efficiency of immunogen development for vaccine therapy (8,(26)(27)(28)(29)(30). We set out to further characterize the binding specificities of these and cancer patient antibodies by using carbohydrate microarray analysis.Carbohydrate microarrays allow for the direct characterization of carbohydrate-protein interactions. In addition, the attachment of sugars to surfaces can effectively mimic the presentation of these compounds on the cell membrane. A large factor which is present...

Highlights d Autoinhibited (3.3 Å ) and active (6.8 Å ) structures of prodegenerative NADase SARM1 solved d Identification of a critical autoinhibitory lock d Lock mutations convert inactive SARM1 to an active, neurotoxic state d Enzymatic studies explain SARM1's functional dependence on local metabolic environment

The Obg family of GTPases is widely conserved and predicted to play an as-yet-unknown role in translation. Recent reports provide circumstantial evidence that both eukaryotic and prokaryotic Obg proteins are associated with the large ribosomal subunit. Here we provide direct evidence that the Caulobacter crescentus CgtA C protein is associated with the free large (50S) ribosomal subunit but not with 70S monosomes or with translating ribosomes. In contrast to the Bacillus subtilis and Escherichia coli proteins, CgtA C does not fractionate in a large complex by gel filtration, indicating a moderately weak association with the 50S subunit. Moreover, binding of CgtA C to the 50S particle is sensitive to salt concentration and buffer composition but not guanine nucleotide occupancy of CgtA C . Assays of epitope-tagged wild-type and mutant variants of CgtA C indicate that the C terminus of CgtA C is critical for 50S association. Interestingly, the addition of a C-terminal epitope tag also affected the ability of various cgtA C alleles to function in vivo. Depletion of CgtA C led to perturbations in the polysome profile, raising the possibility that CgtA C is involved in ribosome assembly or stability.

The Golgi complex is essential for many aspects of cellular function, including trafficking and sorting of membrane and secretory proteins and posttranslational modification by glycosylation. We observed reversible fragmentation of the Golgi complex in cultured hippocampal neurons cultured in hyperexcitable conditions. In addition, Golgi fragmentation was found in cultured neurons with hyperactivity due to prolonged blockade of GABA A -mediated inhibition or withdrawal of NMDA receptor antagonism. The interplay between neuronal hyperactivity and Golgi structure established in this study thus reveals a previously uncharacterized impact of neuronal activity on organelle structure. This finding may have important roles in protein processing and trafficking in the Golgi as well as effects on neuronal signaling.hyperexcitability | activity-dependent

We report the design, synthesis, membrane activity, biophysical characterization, and in vitro antibacterial activities of cationic cyclic D,L-α-glycopeptides bearing side chains derivatized with Dglucosamine (GlcNH 2 ), D-galactose (Gal), or D-mannose (Man).The increasing prevalence of bacterial infections that are resistant to existing antibiotic therapeutics has fueled renewed interest in the design and discovery of novel antibacterial agents. 1 Consequently, a Streptomyces isolate known since the late 1950s to exhibit antibiotic activity against Gram-positive bacteria was recently reexamined, leading to the discovery and structure elucidation in 2002 of a new class of cationic glycopeptide antibiotics named mannopeptimycins. 2 Independently, we had described a new class of rationally designed supramolecular antimicrobial agents based on the self-assembling cyclic D,L-α-peptide architecture developed and studied in our laboratory since early 1990s. [3][4][5] Following the disclosure of the mannopeptimycin structures, we noted that they possess a cyclic peptide architecture, backbone conformation, and potentially membrane-active antibacterial mode of action that are strikingly similar to that of the cyclic D,L-α-peptides. However, unlike mannopeptimycins, the cyclic D,L-α-peptides can be readily synthesized and subjected to multiple rounds of sequence-activity optimizations through either parallel or combinatorial library approaches. 3,4,6 We therefore set out to expand the class of cyclic D,L-α-peptides to include glycosylated analogues and explore the utility of these derivatives as antimicrobial agents. Here we describe initial studies in the design, synthesis, membrane activity, and in vitro antibacterial activity of cationic cyclic D,L-α-glycopeptides bearing side chains derivatized with either D-glucosamine, D-galactose, or D-mannose. 7,8 As a progenitor peptide sequence for the present studies, we chose the membrane-active amphiphilic cyclic D,L-α-peptide [WLWKSKSK] (peptide 8). The symmetrical disposition of hydrophilic amino acids in 8 seemed appropriate for probing rationally the effects of the position and type of O-glycosylserine residue substitutions on antibacterial activity and target membrane selectivity. Accordingly, glycopeptides 1-3 were designed to maintain a similar overall cationic character by individually substituting each of the three lysine residue with serine(βGlcNH 2 ). Similarly, both of the neutral serine residues were individually replaced with either serine(βGal) or serine(αMan), providing glycopeptides 4 and 5 or 6 and 7, respectively (Fig. 1, Table 1).The requisite peracetylated O-linked glycosyl Fmoc-serine derivatives 9, 10, and 11 were synthesized according to published methods 7,9 (Fig. 1). Cyclic peptides 1-8 were synthesized on the solid-support by first tethering Fmoc-D-Lys-OAll via its free ε-amino side chain moiety to a 2-chlorotrityl chloride polystyrene resin. Linear peptide synthesis was carried out according to standard Fmoc synthesis protocols under base-...

A high‐yield one‐pot synthesis of S‐linked glycosyl amino acids 1 has been developed (see scheme; DMF=dimethyl formamide) and used in the solid‐phase synthesis of S‐linked glycopeptides. This approach has been shown to be efficient for the synthesis of various S‐linked glycosyl amino acid building blocks.