Fr Francium

Aligning the haystack to expose the needle: The 3DM method was used to generate a comprehensive database of the α/β-hydrolase fold enzyme superfamily. This database facilitates the analysis of structure–function relationships and enables novel insights into this superfamily to be made. In addition high-quality libraries for protein engineering can be easily designed.

A Fc–PNA biosensor (Fc: ferrocenyl, C10H9Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc–PNA capture probes were therefore synthesized by N-terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi-reversible redox processes of the Fc0/+ redox couple with a significant difference in their electrochemical half-wave potentials of ΔE1/2=160 mV. A carefully designed biosensor interface, consisting of a ternary self-assembled monolayer (SAM) of the two C-terminal cysteine-tethered Fc–PNA capture probes and 6-mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear-cut electrochemical signal about the hybridization event. The discrimination of single-nucleotide polymorphism (SNP) was proven with a chosen single-mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual-potential sensor under real-life conditions.A DNA biosensing interface was designed that uses a binary Fc–PNA recognition layer of two electrochemically distinguishable PNA sequences. Performing single square-wave voltametry measurements, this sensor enables good discrimination of single-nucleotide polymorphism sequences. Preliminary experiments demonstrate that this sensor is compatible with biological samples and has promise for real-life applications.

Small peptides and oligosaccharides are important antigens for the development of vaccines and the production of monoclonal antibodies. Because of their small size, peptides and oligosaccharides are non-immunogenic on their own and typically must be conjugated to a larger carrier protein to elicit an immune response. Selection of a suitable carrier protein, conjugation method, and hapten density are critical for generating an optimal immune response. We used a glycan array to compare the repertoire of antibodies induced after immunizing with either low or high-density conjugates of the tumor-associated Tn antigen. At high hapten density, a broader range of antibodies was induced, and reactivity to the clustered Tn antigen was observed. In contrast, antibodies induced by the low-density conjugate had narrower reactivity and did not bind the clustered Tn antigen.A carbohydrate microarray was used to evaluate the effects of hapten density on the spectrum of antibodies elicited to a tumor-associated carbohydrate antigen. We demonstrate that high hapten density produces a broader range of antibodies and low hapten density induces a narrower range of antibodies.

Transcriptional switch: A zinc-finger protein (CDH2-ZF3) has been created in which a Cys was replaced by an Asp. The ZnII binding affinity of the CDH2-type zinc finger was decreased by ∼500 times compared to wild (C2H2) type. The DNA binding of CDH2-ZF3 could be controlled by the ZnII concentration, and a CDH2-ZF3-based artificial transcription factor clearly showed ZnII-responsive transcriptional activity.

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide-binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp2Glu2 metal-coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide-binding peptide incorporated the following key elements: 1) residues with high α-helix and β-sheet propensities in the respective secondary structures; 2) an optimized big box α-helix N-cap; 3) a Schellman α-helix C-cap motif; and 4) an optional D-Pro-Ser type II’ β-turn in the β-hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25-residue peptide that was a general lanthanide-binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er3+. CD spectra of the peptide-lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N-terminal β-hairpin and the C terminal α-helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D-Pro-Ser type II’ β-turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF-hand peptide.Full-metallo jacket: Lanthanides are versatile metals with unique luminescent and magnetic properties, and they have diverse applications in imaging. A novel, compact, tunable protein-based lanthanide-binding motif was developed using iterative protein design. This work points toward new functional proteins that exploit the chemical possibilities of lanthanides and provides a new way to incorporate lanthanides into proteins.

A ligation strategy based on the Diels–Alder [4+2] cycloaddition for the two-step activity-based labeling of endogenously expressed enzymes in complex biological samples has been developed. A panel of four diene-derivatized proteasome probes was synthesized, along with a dienophile-functionalized BODIPY(TMR) tag. These probes were applied in a Diels–Alder labeling procedure that enabled us to label active proteasome β-subunits selectively in cellular extracts and in living cells. We were also able to label the activity of cysteine proteases in cell extracts by utilizing a diene-derivatized cathepsin probe. Importantly, the Diels–Alder strategy described here is fully orthogonal with respect to the Staudinger–Bertozzi ligation, as demonstrated by the independent labeling of different proteolytic activities by the two methods in a single experiment.Double labeling: A Diels–Alder-based ligation strategy for activity-based profiling of endogenously expressed proteases by using a panel of diene-derivatized probes and a dienophile-functionalized fluorescent tag has been developed. This procedure is fully orthogonal with respect to the Staudinger–Bertozzi ligation, thus allowing both methods to be used in the same sample to independently label two different enzymatic activities.

Hypothemycin and related resorcylic acid lactones (RAL) bearing a cis-enone moiety have emerged as an alternative pharmacophore to heterocyclic motifs for kinase inhibition, and are endowed with a unique selectivity filter based on the irreversible reaction with a subset of the kinome bearing a suitably positioned cysteine residue. Two prototypical examples of “edited” RAL were evaluated for antitumoral, antimetastatic and antiangiogenic efficacy in an orthotopic murine renal cell carcinoma (RENCA) model. Both compounds (3 and 5) are good inhibitors of VEGFRs in vitro, and inhibited tumor growth in vivo with comparable efficacy to sunitinib, an FDA-approved VEGFRs inhibitor. Compound 3 promoted lung metastasis to a similar extent as sunitinib, while compound 5 strongly inhibited lung metastasis. This study attests to the potential of irreversible kinase inhibitors and molecular editing of natural pharmacophores and provides encouraging results to a clinically significant problem.Inspirational kinase inhibitors: The terminal inhibition achieved with irreversible inhibitors has frequently been harnessed by Nature. Resorcyclic acid lactones (RAL) bearing a cis-enone moiety have emerged as promising pharmacophores for kinase inhibition. Two prototypical edited RALs were evaluated on an orthotopic murine carcinoma. Despite their similar biochemical activity profile, remarkable differences were observed with respect to metastasis inhibition.

Chemoenzymatic synthesis: A chemoenzymatic route for modification of aminoglycosides is disclosed. The significant feature of this approach is the discovery that an aminoglycoside 3-N-acetyltransferase, AAC(3)-IV, from Escherichia coli accepts azidoacetyl coenzyme A (AzAcCoA) as a substrate that can be further chemically diversified. This approach was used to develop a carbohydrate microarray method to study modification of aminoglycosides.

Chemical defense of leaf beetle larvae (Chrysomelidae) against enemies is provided by secretions containing a wide range of deterrent compounds or by unpalatable hemolymph constituents. Here we report a new, very strong feeding deterrent against ants released by larvae of the alder leaf beetle Agelastica alni when attacked. The larvae release a defensive fluid from openings of pairwise, dorsolaterally located tubercles on the first to the eighth abdominal segments. The fluid, consisting of hemolymph and probably a glandular cell secretion, has previously been shown to contain a very stable, non-volatile feeding deterrent. The major deterrent component was isolated by repeated HPLC separation and analyzed by NMR and MS. The compound proved to be γ-L-glutamyl-L-2-furylalanine (1), a novel dipeptide containing the unusual amino acid L-2-furylalanine. This amino acid, although synthetically well known, has not previously been reported from natural sources. The absolute configuration of the natural compound was elucidated by enantioselective gas chromatography after derivatization. The structure of the dipeptide was verified by the synthesis of several isomeric dipeptides. In bioassays a concentration of 1 μg μL−1 was sufficient to deter polyphagous Myrmica rubra ants from feeding.Off the menu: We report a new, very strong feeding deterrent against ants released by larvae of the alder leaf beetle Agelastica alni when attacked. The major deterrent component proved to be γ-L-glutamyl-L-2-furylalanine, a novel dipeptide containing the unusual amino acid L-2-furylalanine, which, although synthetically well known, has not previously been reported from natural sources.

Targeting a single cytosine in the presence of others: The deamination coupled with 3-cyanovinylcarbazole-nucleoside-mediated photo-crosslinking allows heat-induced conversion of cytosine into uracil with high site-specificity and without side reactions.