Is it the shape of the cavity, or the shape of the water in the cavity?

Historical interpretations of the thermodynamics characterizing biomolecular recognition have marginalized the role of water. An important (even, perhaps, dominant) contribution to molecular recognition in water comes from the “hydrophobic effect,” in which non-polar portions of a ligand interact preferentially with non-polar regions of a protein. Water surrounds the ligand, and water fills the binding pocket of the protein: when the protein-ligand complex forms, and hydrophobic surfaces of the binding pocket and the ligand approach one another, the molecules (and hydrogen-bonded networks of molecules) of water associated with both surfaces rearrange and, in part, entirely escape into the bulk solution. It is now clear that neither of the two most commonly cited rationalizations for the hydrophobic effect—an entropy-dominated hydrophobic effect, in which ordered waters at the surface of the ligand, and water at the surface of the protein, are released to the bulk upon binding, and a “lock-and-key” model, in which the surface of a ligand interacts directly with a surface of a protein having a complementary shape–can account for water-mediated interactions between the ligand and the protein, and neither is sufficient to account for the experimental observation of both entropy- andenthalpy-dominated hydrophobic effects. What is now clear is that there is no single hydrophobic effect, with a universally applicable, common, thermodynamic description: different processes (i.e., partitioning between phases of different hydrophobicity, aggregation in water, and binding) with different thermodynamics, depend on the molecular-level details of the structures of the molecules involved, and of the aggregates that form. A “water-centric” description of the hydrophobic effect in biomolecular recognition focuses on the structures of water surrounding the ligand, and of water filling the binding pocket of the protein, both before and after binding. This view attributes the hydrophobic effect to changes in the free energy of the networks of hydrogen bonds that are formed, broken, or re-arranged when two hydrophobic surfaces approach (but do not necessarily contact) one another. The details of the molecular topography (and the polar character) of the mole- cular surfaces play an important role in determining the structure of these networks of hydrogen-bonded waters, and in the thermodynamic description of the hydrophobic effect(s). Theorists have led the formulation of this “water-centric view”, although experiments are now supplying support for it. It poses complex problems for would-be “designers” of protein-ligand interactions, and for so-called “rational drug design”.Chemistry and Chemical Biolog

Analyzing Forensic Evidence Based on Density with Magnetic Levitation

This paper describes a method for determining the density of contact trace objects with magnetic levitation (MagLev). MagLev measurements accurately determine the density (±0.0002 g/cm3) of a diamagnetic object and are compatible with objects that are nonuniform in shape and size. The MagLev device (composed of two permanent magnets with like poles facing) and the method described provide a means of accurately determining the density of trace objects. This method is inexpensive, rapid, and verifiable and provides numerical values—independent of the specific apparatus or analyst—that correspond to the absolute density of the sample that may be entered into a searchable database. We discuss the feasibility of MagLev as a possible means of characterizing forensic-related evidence and demonstrate the ability of MagLev to (i) determine the density of samples of glitter and gunpowder, (ii) separate glitter particles of different densities, and (iii) determine the density of a glitter sample that was removed from a complex sample matrix.Chemistry and Chemical Biolog

\u3ci\u3eIn situ\u3c/i\u3e Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the reactive layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA). Postfabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization/dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multistep synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ∼450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents

Selective Precipitation and Purification of Monovalent Proteins Using Oligovalent Ligands and Ammonium Sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein–ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein–ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins—for which appropriate oligovalent ligands can be synthesized—and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.Chemistry and Chemical Biolog

The Binding of Benzoarylsulfonamide Ligands to Human Carbonic Anhydrase is Insensitive to Formal Fluorination of the Ligand

Auf das Wasser kommt es an: Konservierte Bindungsgeometrie, enthalpiegetriebene Bindungsweise und nichtunterscheidbare Bindungsaffinitäten für fluorierte und nichtfluorierte Liganden von humaner Carbonsäureanhydrase (siehe Bild) stützen die Theorie, dass das Schlüssel-Schloss-Modell einen wichtigen Bestandteil des Bindungsvorgangs unbeachtet lässt: das Wasser, das die Bindungstasche des Proteins füllt und den Liganden umgibt.Chemistry and Chemical Biolog

Filter-Based Assay for Escherichia coli in Aqueous Samples Using Bacteriophage-Based Amplification

This paper describes a method to detect the presence of bacteria in aqueous samples, based on the capture of bacteria on a syringe filter, and the infection of targeted bacterial species with a bacteriophage (phage). The use of phage as a reagent provides two opportunities for signal amplification: i) the replication of phage inside a live bacterial host (1000-fold amplification for M13 phage in E. coli K12), and ii) the rapid conversion of a colorless substrate to a colored or fluorescent product by an enzyme that is co-expressed with the phage (in this demonstration β- galactosidase, which has a turnover rate of ~ 600 molecules/second). This method can detect a single colony-forming unit (CFU) of E. coli in one liter of water with an overnight culture-based assay, or 50 CFUs of E. coli in 1 liter of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than four hours with a solution-based assay with visual readout. The solution-based assay does not require specialized equipment or access to a laboratory, and is more rapid than existing tests that are suitable for use at the point of access. This method could be applied to the detection of many different bacteria, in parallel, with bacteriophages that express enzymes not natively expressed in the target bacteria.Chemistry and Chemical Biolog

Rectification in Tunneling Junctions: 2,2′-Bipyridyl-Terminated n -Alkanethiolates

Molecular rectification is a particularly attractive phenomenon to examine in studying structure–property relationships in charge transport across molecular junctions, since the tunneling currents across the same molecular junction are measured, with only a change in the sign of the bias, with the same electrodes, molecule(s), and contacts. This type of experiment minimizes the complexities arising from measurements of current densities at one polarity using replicate junctions. This paper describes a new organic molecular rectifier: a junction having the structure AgTS/S(CH2)11-4-methyl-2,2′-bipyridyl//Ga2O3/EGaIn (AgTS: template-stripped silver substrate; EGaIn: eutectic gallium–indium alloy) which shows reproducible rectification with a mean r+ = |J(+1.0 V)|/|J(−1.0 V)| = 85 ± 2. This system is important because rectification occurs at a polarity opposite to that of the analogous but much more extensively studied systems based on ferrocene. It establishes (again) that rectification is due to the SAM, and not to redox reactions involving the Ga2O3 film, and confirms that rectification is not related to the polarity in the junction. Comparisons among SAM-based junctions incorporating the Ga2O3/EGaIn top electrode and a variety of heterocyclic terminal groups indicate that the metal-free bipyridyl group, not other features of the junction, is responsible for the rectification. The paper also describes a structural and mechanistic hypothesis that suggests a partial rationalization of values of rectification available in the literature.Chemistry and Chemical Biolog