Fr Francium

The substrate mimetics approach is a versatile method for small-scale enzymatic peptide-bond synthesis in aqueous systems. The protease-recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z-X_AA-OGp esters, computational docking studies were performed. The results strongly point at enzyme-specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain.

Fooling with substrate recognition: Guanidinophenyl (OGp) esters were utilized as potential substrate mimetics for papain-induced dipeptide synthesis under aqueous conditions. Surprisingly, modeling studies instead revealed unprecedented enzyme-specific activation, applicable to nearly all amino acids.

Fatty acyl-AMP ligases (FAALs) activate fatty acids as acyladenylates, and subsequently catalyze their transfer onto the acyl carrier proteins (ACPs) of polyketide synthases (PKSs) or nonribosomal peptide synthetases to produce lipidic metabolites. Myxococcus xanthus contains a polyketide biosynthesis gene cluster in which putative FAAL (FtpD) and ACP (FtpC) genes are located close to a type III PKS (FtpA) gene. Here we describe the characterization of these three proteins in vitro. FtpD adenylated stearic acid and produced stearoyl-FtpC. The stearoyl moiety was then transferred to FtpA. When extender substrates (malonyl-CoA and methylmalonyl-CoA) were added to the reaction, the alkylresorcinol 5-heptadecyl-4-methyl-benzene-1,3-diol was synthesized. Further in vitro analysis indicated that FtpA produces an alkylresorcylic acid as the direct product, and that this decarboxylates to alkylresorcinol nonenzymatically. This is the first report of a FAAL supplying a long-chain fatty acyl-ACP starter substrate to a type III PKS.

Chewing the fat:Myxococcus xanthus contains a polyketide biosynthesis gene cluster, in which putative FAAL and ACP genes are located in close proximity to a type III PKS gene. In vitro characterization of these three proteins revealed that FAAL produces a long-chain fatty acyl-ACP and supplies it to the type III PKS as a starter substrate for alkylresorcylic acid production.

5-Ethynyl-2′-deoxycytidine triphosphate (EdCTP) was synthesized as a probe to be used in conjunction with fluorescent labeling to facilitate the analysis of the in vivo dynamics of DNA-centered processes (DNA replication, repair and cytosine demethylation). Kinetic analysis showed that EdCTP is accepted as a substrate by Klenow exo⁻ and DNA polymerase β. Incorporation of 5-ethynyl-2′-deoxycytidine (EdC) into DNA by these enzymes is, at most, modestly less efficient than native dC. EdC-containing DNA was visualized by using a click reaction with a fluorescent azide, following polymerase incorporation and T4 DNA ligase mediated ligation. Subsequent experiments in mouse male germ cells and zygotes demonstrated that EdC is a specific and reliable reporter of DNA replication, in vivo.

Tracking cytosine incorporation: 5-Ethynyl-2′-deoxycytidine (EdC; see figure) was synthesized and validated for tracking DNA-centered processes in live mouse male germ cells and one-cell embryos. Using this technique we show that EdC can be used as a mechanism-based tool to further explore the chemical mechanism of DNA demethylation.

Latent thioesters: Reverse native chemical ligation followed by intramolecular trans-thioesterification and acyl transfer can rearrange linear peptides into biologically active cyclic products.

The 3D structures of six linear pentadecapeptides derived from the cecropin A–melittin antimicrobial peptide CA(1–7)M(2–9) [KWKLFKKIGAVLKVL-NH₂] have been studied. These analogues are modified by ε-NH₂ trimethylation of one or more lysine residues and showed variation in both antimicrobial and cytotoxic activities, depending on the number and position of modified lysines. Since it is expected that these peptides will display a strong conformational ordering when in contact with membranes, we have investigated their structure on the basis of the data extracted from NMR experiments performed in membrane-mimetic environments. We show that inclusion of N^ε-trimethylated lysine residues induces a certain degree of structural flexibility, while preserving to a variable extent a largely α-helical structure. In addition, peptide orientation with respect to SDS micelles has been explored by detection of the intensity changes of peptide NMR signals upon addition of a paramagnetic probe (Mn²⁺ ions).

Tuning antimicrobial activity: Conformational studies of a potent synthetic antimicrobial hybrid peptide and a set of its analogues have been carried out in membrane-mimetic media. Selective substitutions of lysine residues by N^ε-trimethylated lysines notably alter its antihemolytic activity although they have little structural impact.

Selection of a larger pair: Nucleic acid duplexes with non-Watson–Crick base pairs are of interest to fields that span structural biology, supramolecular chemistry, origin of life, and synthetic biology. Here we demonstrate that intercalating molecules can exhibit selective binding of duplexes with purine–purine base pairs over duplexes with Watson–Crick base pairs, and vice versa.

Currently no structurally sensitive spectroscopic techniques are capable of co-determining ensemble structural content and localized lipid versus aqueous solvation information. Here, we describe the first deep-UV (λ_ex<210 nm) resonance Raman (dUVRR) spectra of a model α-helical peptide embedded in a membrane-mimetic environment, confirming sensitivity to secondary structure content and revealing sensitivity of dUVRR to the lipid solvation of the peptide backbone.

The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O⁶-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged β-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP_f, which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP_f and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.

A cleaner image: We report the design and application of an improved labeling system, which combines the use of a faster reacting variant of SNAP-tag, termed SNAP_f, with fluorogenic benzylguanine probes for wash-free labeling of fusion proteins in living cells.

Maintaining your integrity: Morphologically friendly DESI-MS imaging of tissue can be performed prior to histochemical and immuno-histochemical analysis on same tissue section by using new solvent systems. This novel DESI-MS imaging capability allows chemical information to be obtained while preserving tissue integrity.

Membrane potential indifference: A recent cell-based, high-throughput screening of a large aromatic group chemical library discovered a potential fluorescent probe that is able to stain mitochondria in living cells. Structural analyses indicate that the selected molecule becomes fluorescent within the cells through bioconversion with high specificity for mitochondrial surfaces.