Detection of phosphoproteins on electroblot membranes using a small-molecule organic fluorophore

A new formulation of the small-molecule organic fluorophore, Pro-Q Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.     

An aptazyme-based molecular device that converts a small-molecule input into an RNA output

We describe the construction of an aptazyme-based molecular device that converts, through a cascade of reactions, a small-molecule input into output RNA strands. This device is applicable as an interface between a small molecule and a molecular system that accepts only nucleic acid input.     

Fully automated liquid extraction‐based surface sampling and ionization using a chip‐based robotic nanoelectrospray platform

A fully automated liquid extraction‐based surface sampling device utilizing an Advion NanoMate chip‐based infusion nanoelectrospray ionization system is reported. Analyses were enabled for discrete spot sampling by using the Advanced User Interface of the current commercial control software. This software interface provided the parameter control necessary for the NanoMate robotic pipettor to both form and withdraw a liquid microjunction for sampling from a surface. The system was tested with three types of analytically important sample surface types, viz., spotted sample arrays on a MALDI plate, dried blood spots on paper, and whole‐body thin tissue sections from drug dosed mice. The qualitative and quantitative data were consistent with previous studies employing other liquid extraction‐based surface sampling techniques. Published in 2009 by John Wiley & Sons, Ltd.     

Energetics of ion transport in a peptide nanotube

Ion channels constitute an important family of integral membrane proteins responsible for the regulation of ion transport across the cell membrane. Yet, the underlying energetics of the permeation events and how the latter are modulated by the environment, specifically near the mouth of the pore, remain only partially characterized. Here, a synthetic membrane channel formed by cyclic peptides of alternated d- and l-hydrophobic alpha-amino acids was considered. The free energy delineating the translocation of a sodium ion was measured along the conduction pathway by means of molecular dynamics simulations. The free-energy profiles that underly the permeation of the open-ended tubular structure are shown to not only depend on the characteristics of the latter but also inherently on the location of the mouth of the synthetic channel with respect to the membrane surface.     

Electron transport in a pi-stacking molecular chain

We have investigated the electronic structure and transport properties of a pi-stacking molecular chain which is covalently bonded to a H/Si(100) surface, using the first-principles density functional theory approach combined with Green's function method. The highest occupied molecular orbital (HOMO) dispersion is remarkably reduced, but remains noticeable ( approximately 0.1 eV), when a short pi-stacking styrene wire is cut from an infinitely long wire and sandwiched between metal electrodes. We find that the styrene chain's HOMO and lowest unoccupied molecular orbital (LUMO) states are not separated from Si, indicating that it does not work as a wire. By substituting -NO2 or -NH2 for the top -H of styrene, we are able to shift the position of the HOMO and LUMO with respect to the Fermi level. More importantly, we find that the HOMO of styrene-NH2 falls into the band gap of the substrate and is localized in the pi-stacking chain, which is what we need for a wire to be electrically separated from the substrate. The conductance of such an assembly is comparable to that of Au/benzene dithiolate/Au wire based on chemical bonding, and its tunability makes it a promising system for a molecular device.     

Analytical results on ballistic transport in a periodic molecular wire

In this paper, we study the coherent electronic transport of a periodic quantum wire (P-QW) such as poly-acetylene connected to uniform metallic leads, within the tight-binding (TB) approach and in the ballistic regime. We have calculated the Green’s function (GF), density of states (DOS) and the coherent transmission coefficient (TC) fully exactly for a quantum wire. The quasi gap and the energy and wire-length dependence of the GF and conductance for the system are also derived. Finally, we obtain a non-linear equation which gives the bound state energies. Our calculation can be generalized to arbitrary leads and can be applied to molecular wires, polymers, nanocrystals, where results may be useful in designing future molecular electronic devices.     

Study of exciton transport in luminescent molecular nanoclusters using energy traps

We used a squarylium dye SQ as a specific exciton trap for J aggregates of the amphiphilic cyanine dye amphi-PIC. The exciton transport parameters in amphi-PIC J aggregates were estimated using a modified Stern–Volmer equation. We found that SQ quenches 50% of the luminescence of amphi-PIC J aggregates for a ratio of 1 SQ molecule per 80 amphi-PIC molecules. Translated from Teoreticheskaya i éksperimental’naya Khimiya, Vol. 45, No. 1, pp. 50–53, January–February, 2009.     

Construction of a peptide with an electroactive daunomycin like a pendant arm to detect ovalbumin

In this study, a peptide-1 (RNRCKGTDVQAW) constructing lysozyme was conjugated with an electroactive daunomycin in order to voltammetrically detect ovalbumin (OVA). Hetero-bifunctional cross-linking agents with four kinds of ethylene chains in differing lengths were used to bind the peptide-1 and daunomycin. After a cross-linking agent had reacted with an amino group of daunomycin, the compound was introduced into the peptide to the cysteine residue in the peptide using a pendant arm. The OVA was sensed via a change in the electrode response of the daunomycin moiety, based on the binding between the peptide and the OVA. The adsorption of the peptide probe on the electrode increased with increases in the ethylene chain. The binding constants between the peptide probes and the OVA, however, did not depend on the length of the chain. This was because the ethylene chain influenced the binding. When the peptide and the daunomycin were bound using N-(6-maleimidocaproyloxy) sulfosuccinimide, the electrode response of the peptide probe was the most sensitive from among the four cross-linking agents. The calibration curve of the OVA using the peptide probe was linear and ranged from 1.5 × 10⁻¹¹ to 3.0 × 10⁻¹⁰ M. Furthermore, this method could be applied to the electrochemical sensing of the OVA in egg whites and in fetal bovine serum.     

Electron transport through molecular wires based on a face-shared bioctahedral motif

Density functional theory in conjunction with non-equilibrium Green's functions is used to explore the electron transport properties of a series of molecules based on the face-shared bioctahedral (M₂Cl₉) motif. The metal-metal bond orders in the chosen molecules, [Rh₂Cl₉]^3–, [Ru₂Cl₉]^3– and [Mo₂Cl₉]^3– vary from 0 (Rh) to 1 (Ru) and 3 (Mo), and the calculations indicate that there is a direct correlation between conductance and bond order. The [Mo₂Cl₉]^3– case is particularly interesting as it is well known from crystallographic studies to be very flexible, the Mo–Mo bond length varying over a range of ～0.35 Å depending on cation. The upper limit of this range marks the point where homolytic cleavage of the δ_π components of the triple bond is complete, and this has a marked impact on electron transport. The localization of the metal-based orbitals means that those on the left (source) and right (drain) sides respond very differently to applied bias, giving rise to resonance effects at particular bias voltages, and hence to negative differential resistance effects.     

Prospects in computational molecular medicine: a millennial mega-project on peptide folding

During the second half of the 20th century, Molecular Computations have reached to a level that can revolutionize chemistry. The next target will be structural biology, which will be followed soon by Molecular Medicine. The present paper outlines where we are at, in this field, at the end of the 20th century, and in what direction the development may take in the new millennium. In view of the gigantic nature of the problem, it is suggested that a suitably designed cooperative Millennial Mega-project might accelerate our schedule.     

Examination of the folding of a short alanine-based helical peptide with salt bridges using molecular dynamics simulation

A molecular dynamics simulation of the folding of a short alanine-based helical peptide of 17 residues with three Glu...Lys (i, i + 4) salt bridge pairs, referred to as the AEK17 peptide, was carried out. The simulation gave an estimated simulation folding time of 2.5 ns, shorter than 12 ns for an alanine-based peptide of 16 residues with three Lys residues only, referred to as the AK16 peptide, simulated previously. After folded, the AEK17 peptide had a helical content of 77%, in excellent agreement with the experimentally determined value of 80%. An examination of the folding pathways of AEK17 indicated that the peptide proceeded via three-turn helix conformations more than the helix-turn-helix conformation in the folding pathways. An analysis of interactions indicated that the formation of hydrogen bonds between Lys residue side chains and backbone carbonyls is a major factor in the abundant conformation of the three-turn helix intermediate. The substitution of three Ala with Glu residues reduces the extent of hydrophobic interaction in alanine-based AK peptides with the result that the breaking of the interactions of Lys epsilon-NH3+(side chain)...C=O(backbone) is a major activation action for the AEK17 to achieve a complete fold, in contrast to the AK16 peptide, in which breaking non-native hydrophobic interaction is the rate-determining step.     

Fluorescent dendron-cyclodextrin nanotubes with surface peptide spacer as a recyclable sensory platform

Nanotube sensory platform: Dendron-cyclodextrin nanotubes with a surface coumarin unit attached by a GH dipeptide spacer were constructed by a combination of molecular recognition and self-assembly. These unique fluorescent nanotubes can serve as a recyclable metal ion sensory platform with high selectivity and sensitivity (see scheme).     

One-step labelling of a novel small-molecule peptide with astatine-211: preliminary evaluation in vitro and in vivo

In this paper, VP2, a novel small molecule fusion peptide, was labelled with ²¹¹At through a one-step method (coupled with bifunctional intermediate SPC first and then labelled) with a radiochemical yield of about 45%. The radiochemical purity was still > 95% after 24 h at room temperature. Specificity studies in vitro indicated that ²¹¹At-SPC-VP2 has a high affinity for several tumour cells. Additionally, biodistribution in KM mice showed that ²¹¹At-SPC-VP2 has sufficient stability in vivo. This research suggested that ²¹¹At–SPC-VP2 produced by the convenient method has the potential to be a novel targeted drug for cancer radiotherapy.     

Recent advances on small-molecule fluorophores with emission beyond 1000 nm for better molecular imaging in vivo

Small-molecule NIR-II fluorophores play prominent roles in NIR-II biomedical research due to their promising properties such as high biocompatibility, fast excretion, favorable pharmacokinetics as well as easy and robust preparation. In order to promote the future translational practice, it is essential to design and synthesize highperformance NIR-II fluorophores.     

Structural flexibility of a helical peptide regulates vibrational energy transport properties

Applying ultrafast vibrational spectroscopy, we find that vibrational energy transport along a helical peptide changes from inefficient but mostly ballistic below approximately 270 K into diffusive and significantly more efficient above. On the basis of molecular dynamics simulations, we attribute this change to the increasing flexibility of the helix above this temperature, similar to the glass transition in proteins. Structural flexibility enhances intramolecular vibrational energy redistribution, thereby refeeding energy into the few vibrational modes that delocalize over large parts of the structure and therefore transport energy efficiently. The paper outlines concepts how one might regulate vibrational energy transport properties in ultrafast photobiological processes, as well as in molecular electronic devices, by engineering the flexibility of their components.     

Electrical transport measurements on self-assembled organic molecular wires

The electrical properties of supermolecular assemblies of oligo(p-phenylene vinylene) were studied. These materials self-assemble into well-defined cylindrical structures in solution with lengths in the range of 100 nm-10 microm and diameters between 5 and 200 nm. Atomic force microscopy showed that by adjusting the concentration, either individual molecular wires or a dense film could be deposited. The molecular wires showed poor electrical conduction. Several tests were performed that show that it was the molecular wires themselves, not the contacts, that limit the conductivity.