Intrinsic energy landscapes of amino acid side-chains

Amino acid side-chain conformational properties influence the overall structural and dynamic properties of proteins and, therefore, their biological functions. In this study, quantum mechanical (QM) potential energy surfaces for the rotation of side-chain χ(1) and χ(2) torsions in dipeptides in the alphaR, beta, and alphaL backbone conformations were calculated. The QM energy surfaces provide a broad view of the intrinsic conformational properties of each amino acid side-chain. The extent to which intrinsic energetics dictates side-chain orientation was studied through comparisons of the QM energy surfaces with χ(1) and χ(2) free energy surfaces from probability distributions obtained from a survey of high resolution crystal structures. In general, the survey probability maxima are centered in minima of the QM surfaces as expected for sp(3) (or sp(2) for χ(2) of Asn, Phe, Trp, and Tyr) atom centers with strong variations between amino acids occurring in the energies of the minima indicating intrinsic differences in rotamer preferences. High correlations between the QM and survey data were found for hydrophobic side-chains except Met, suggesting minimal influence of the protein and solution environments on their conformational distributions. Conversely, low correlations for polar or charged side-chains indicate a dominant role of the environment in stabilizing conformations that are not intrinsically favored. Data also link the presence of off-rotamers in His and Trp to favorable interactions with the backbone. Results also suggest that the intrinsic energetics of the side-chains of Phe and Tyr may play important roles in protein folding and stability. Analyses on whether intrinsic side-chain energetics can influence backbone preference identified a strong correlation for residues in the alphaL backbone conformation. It is suggested that this correlation reflects the intrinsic instability of the alphaL backbone such that assumption of this backbone conformation is facilitated by intrinsically favorable side-chain conformations. Together our results offer a broad overview of the conformational properties of amino acid side-chains and the QM data may be used as target data for force field optimization.     

A photoelectron spectroscopy and ab initio study of B21-: negatively charged boron clusters continue to be planar at 21

The structures and chemical bonding of the B(21)(-) cluster have been investigated by a combined photoelectron spectroscopy and ab initio study. The photoelectron spectrum at 193 nm revealed a very high adiabatic electron binding energy of 4.38 eV for B(21)(-) and a congested spectral pattern. Extensive global minimum searches were conducted using two different methods, followed by high-level calculations of the low-lying isomers. The global minimum of B(21)(-) was found to be a quasiplanar structure with the next low-lying planar isomer only 1.9 kcal/mol higher in energy at the CCSD(T)/6-311-G* level of theory. The calculated vertical detachment energies for the two isomers were found to be in good agreement with the experimental spectrum, suggesting that they were both present experimentally and contributed to the observed spectrum. Chemical bonding analyses showed that both isomers consist of a 14-atom periphery, which is bonded by classical two-center two-electron bonds, and seven interior atoms in the planar structures. A localized two-center two-electron bond is found in the interior of the two planar isomers, in addition to delocalized multi-center σ and π bonds. The structures and the delocalized bonding of the two lowest lying isomers of B(21)(-) were found to be similar to those in the two lowest energy isomers in B(19)(-).     

Efficient electroformation of supergiant unilamellar vesicles containing cationic lipids on ITO-coated electrodes

Giant unilamellar vesicles (GUVs) represent a versatile in vitro system widely used to study properties of lipid membranes and their interaction with biomacromolecules and colloids. Electroformation with indium tin oxide (ITO) coated coverslips as electrodes is a standard approach to GUV production. In the case of cationic GUVs, however, application of this approach leads to notorious difficulties. We discover that this is related to aging of ITO-coated coverslips during their repeated use, which is reflected in their surface topography on the nanoscale. We find that mild annealing of the ITO-coated surface in air reverts the effects of aging and ensures efficient reproducible electroformation of supergiant (diameter > 100 μm) unilamellar vesicles containing cationic lipids.     

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 μL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.     

Degradable conjugates from oxanorbornadiene reagents

Oxanorbornadienedicarboxylate (OND) reagents were explored for purposes of binding and releasing drugs from serum albumins as representative macromolecular carriers. Being highly reactive Michael acceptors, ONDs form adducts with thiols and amines, which then undergo retro-Diels-Alder fragmentation. A study of more than 30 model adducts revealed a number of modifications that can be used to influence adduct stability. For the most reactive OND linkers, the labeling of the single available bovine serum albumin (BSA) cysteine residue was complete within minutes at a mid-micromolar concentration of reactants. While a selectivity of greater than 1000-fold for thiol over amine was observed with model amino acids, the labeling of protein amines with ONDs is fast enough to be practical, as demonstrated by the reaction with thiol-depleted BSA. The OND-amine adducts were found to be up to 15 times more stable than OND-thiol adducts, and to be sensitive to acid by virtue of a stereochemically dependent acceleration of cycloreversion. The release rate of fluorescent cargo from serum albumins was tuned by selecting the coupling partners: the available half-lives ranged from 40 min to 7 days at 37 °C. Such versatility of release profiles from protein carriers, controlled by the nature of the OND linkage, is a useful addition to the drug delivery toolbox.     

Monolithic columns with optimized pore structure for molecular size-based separations of synthetic polymers

Monolithic capillary columns based on divinylbenzene were synthesized using different alcanols as porogens. Prepared columns were tested in separation of polystyrene standards according to their molar mass (MM) and were characterized by corresponding calibration graphs. It was demonstrated that a decrease of alcanol chain length from dodecanol to octanol resulted in a decrease of column permeability and in an improved column ability to separate polystyrene standards. In contrast, removing a good solvent from porogen mixture results in an increase of column permeability and in a decrease of column performance toward polystyrene standards. Optimized synthetic conditions included porogen composed of nonanol and toluene or mesytilene, and the column prepared with this porogen was capable of separating a mixture of 14 polystyrene standards with MM ranged from several millions to oligomers.     

Traceless chemical ligations from O-acyl serine sites

Chemical ligation via O- to N-acyl transfer of O-acylated serine containing peptides affords serine containing native peptides via 8- and 11-membered cyclic transition states opening the door to a wide variety of potential applications to peptide elaboration. The feasibility of these traceless chemical ligations is feasible as supported by computation.     

Spin-orbit quenching of Cl(2P(1∕2)) by H2

We report fully-quantum, time-independent, scattering calculations for the spin-orbit quenching of Cl((2)P(1∕2)) by H(2) molecules at low and moderate temperature. Our calculations take into account chemical reaction channels. Cross sections are calculated for total energies up to 5000 cm(-1) which are used to determine, by thermal averaging, state-to-state rate coefficients at temperatures ranging from 50 to 500 K. Spin-orbit relaxation of chlorine is dominated by collisions with H(2) in the rotationally excited states j = 2 and j = 3. In the former case the near-resonant energy transfer is the primary relaxation mechanism. The inclusion of the reactive channel could lead differences compared to pure inelastic calculations. Good agreement is obtained with experimental relaxation measurements at room temperature.     

In situ synthesis of Cu-BTC (HKUST-1) in macro-/mesoporous silica monoliths for continuous flow catalysis

The metal-organic framework Cu-BTC has been successfully synthesized as nanoparticles inside the mesopores of silica monoliths featuring a homogeneous macropore network enabling the use of Cu-BTC for continuous flow applications in liquid phase with low pressure drop. High productivity was reached with this catalyst for the Friedl?nder reaction.     

Psoromic acid is a selective and covalent Rab-prenylation inhibitor targeting autoinhibited RabGGTase

Post-translational attachment of geranylgeranyl isoprenoids to Rab GTPases, the key organizers of intracellular vesicular transport, is essential for their function. Rab geranylgeranyl transferase (RabGGTase) is responsible for prenylation of Rab proteins. Recently, RabGGTase inhibitors have been proposed to be potential therapeutics for treatment of cancer and osteoporosis. However, the development of RabGGTase selective inhibitors is complicated by its structural and functional similarity to other protein prenyltransferases. Herein we report identification of the natural product psoromic acid (PA) that potently and selectively inhibits RabGGTase with an IC(50) of 1.3 μM. Structure-activity relationship analysis suggested a minimal structure involving the depsidone core with a 3-hydroxyl and 4-aldehyde motif for binding to RabGGTase. Analysis of the crystal structure of the RabGGTase:PA complex revealed that PA forms largely hydrophobic interactions with the isoprenoid binding site of RabGGTase and that it attaches covalently to the N-terminus of the α subunit. We found that in contrast to other protein prenyltransferases, RabGGTase is autoinhibited through N-terminal (α)His2 coordination with the catalytic zinc ion. Mutation of (α)His dramatically enhances the reaction rate, indicating that the activity of RabGGTase is likely regulated in vivo. The covalent binding of PA to the N-terminus of the RabGGTase α subunit seems to potentiate its interaction with the active site and explains the selectivity of PA for RabGGTase. Therefore, psoromic acid provides a new starting point for the development of selective RabGGTase inhibitors.