Matthew R. Lockett

Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase

The hydrophobic effect—a rationalization of the insolubility of nonpolar molecules in water—is centrally important to biomolecular recognition. Despite extensive research devoted to the hydrophobic effect, its molecular mechanisms remain controversial, and there are still no reliably predictive models for its role in protein–ligand binding. Here we describe a particularly well-defined system of protein and ligands—carbonic anhydrase and a series of structurally homologous heterocyclic aromatic sulfonamides—that we use to characterize hydrophobic interactions thermodynamically and structurally. In binding to this structurally rigid protein, a set of ligands (also defined to be structurally rigid) shows the expected gain in binding free energy as hydrophobic surface area is added. Isothermal titration calorimetry demonstrates that enthalpy determines these increases in binding affinity, and that changes in the heat capacity of binding are negative. X-ray crystallography and molecular dynamics simulations are compatible with the proposal that the differences in binding between the homologous ligands stem from changes in the number and organization of water molecules localized in the active site in the bound complexes, rather than (or perhaps in addition to) release of structured water from the apposed hydrophobic surfaces. These results support the hypothesis that structured water molecules—including both the molecules of water displaced by the ligands and those reorganized upon ligand binding—determine the thermodynamics of binding of these ligands at the active site of the protein. Hydrophobic effects in various contexts have different structural and thermodynamic origins, although all may be manifestations of the differences in characteristics of bulk water and water close to hydrophobic surfaces.

Water Networks Contribute to Enthalpy/Entropy Compensation in Protein–Ligand Binding

The mechanism (or mechanisms) of enthalpy-entropy (H/S) compensation in protein-ligand binding remains controversial, and there are still no predictive models (theoretical or experimental) in which hypotheses of ligand binding can be readily tested. Here we describe a particularly well-defined system of protein and ligands--human carbonic anhydrase (HCA) and a series of benzothiazole sulfonamide ligands with different patterns of fluorination--that we use to define enthalpy/entropy (H/S) compensation in this system thermodynamically and structurally. The binding affinities of these ligands (with the exception of one ligand, in which the deviation is understood) to HCA are, despite differences in fluorination pattern, indistinguishable; they nonetheless reflect significant and compensating changes in enthalpy and entropy of binding. Analysis reveals that differences in the structure and thermodynamic properties of the waters surrounding the bound ligands are an important contributor to the observed H/S compensation. These results support the hypothesis that the molecules of water filling the active site of a protein, and surrounding the ligand, are as important as the contact interactions between the protein and the ligand for biomolecular recognition, and in determining the thermodynamics of binding.

A paper-based invasion assay: assessing chemotaxis of cancer cells in gradients of oxygen.

This work describes a 3D, paper-based assay that can isolate sub-populations of cells based on their invasiveness (i.e., distance migrated in a hydrogel) in a gradient of concentration of oxygen (O2). Layers of paper impregnated with a cell-compatible hydrogel are stacked and placed in a plastic holder to form the invasion assay. In most assays, the stack comprises a single layer of paper containing mammalian cells suspended in a hydrogel, sandwiched between multiple layers of paper containing only hydrogel. Cells in the stack consume and produce small molecules; these molecules diffuse throughout the stack to generate gradients in the stack, and between the stack and the bulk culture medium. Placing the cell-containing layer in different positions of the stack, or modifying the permeability of the holder to oxygen or proteins, alters the profile of the gradients within the stack. Physically separating the layers after culture isolates sub-populations of cells that migrated different distances, and enables their subsequent analysis or culture. Using this system, three independent cell lines derived from A549 cancer cells are shown to produce distinguishable migration behavior in a gradient of oxygen. This result is the first experimental demonstration that oxygen acts as a chemoattractant for cancer cells.

Hydroxycarboxylic Acid-Derived Organosulfates: Synthesis, Stability, and Quantification in Ambient Aerosol

Organosulfates have been proposed as contributors to aerosol growth and have been detected in both chamber and atmospheric aerosol samples. We present a simple method for the synthesis of quantitative analytical standards of two small hydroxycarboxylic acid-derived organosulfates, glycolic and lactic acid sulfate. Additionally, we discuss the stability of hydroxycarboxylic acid-derived organosulfates and their previously proposed sulfate hemiacetal isomers in commonly used solvents for filter extraction. The hydroxycarboxylic acid-derived organosulfates were found to be stable under acidic conditions comparable to those found in ambient aerosol. By using synthesized standards, quantitative organosulfate concentrations were measured from ambient particulate matter (PM(2.5)) collected in urban locations in the United States, Mexico City, and Pakistan. Lactic acid sulfate and glycolic acid sulfate concentrations ranged 0.4-3.8 ng/m(3) and 1.9-11.3 ng/m(3), respectively. We propose that glycolic acid sulfate represents an important tracer for atmospheric processes that form organosulfates in ambient particulate matter.

Three-dimensional paper-based model for cardiac ischemia.

In vitro models of ischemia have not historically recapitulated the cellular interactions and gradients of molecules that occur in a 3D tissue. This work demonstrates a paper-based 3D culture system that mimics some of the interactions that occur among populations of cells in the heart during ischemia. Multiple layers of paper containing cells, suspended in hydrogels, are stacked to form a layered 3D model of a tissue. Mass transport of oxygen and glucose into this 3D system can be modulated to induce an ischemic environment in the bottom layers of the stack. This ischemic stress induces cardiomyocytes at the bottom of the stack to secrete chemokines which subsequently trigger fibroblasts residing in adjacent layers to migrate toward the ischemic region. This work demonstrates the usefulness of patterned, stacked paper for performing in vitro mechanistic studies of cellular motility and viability within a model of the laminar ventricle tissue of the heart.

Carbon-on-metal films for surface plasmon resonance detection of DNA arrays.

Surface plasmon resonance (SPR) imaging affords label-free monitoring of biomolecule interactions in an array format. A surface plasmon conducting metal thin film is required for SPR measurements. Gold thin films are traditionally used in SPR experiments as they are readily functionalized with thiol-containing molecules through formation of a gold-sulfur bond. The lability of this gold-thiol linkage upon exposure to oxidizing conditions and ultraviolet light renders these surfaces incompatible with light-directed synthetic methods for fabricating DNA arrays. It is shown here that applying a thin carbon overlayer to the gold surface yields a chemically robust substrate that permits light-directed synthesis and also supports surface plasmons. DNA arrays fabricated on these carbon-metal substrates are used to analyze two classes of biomolecular interactions: DNA-DNA and DNA-protein. This new strategy allows the combinatorial study of binding interactions directly from native, unmodified biomolecules of interest and offers the possibility of discovering new ligands in complex mixtures such as cell lysates.

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

Its the water that matters. Pairs of benzo- and perfluorobenzoarylsulfonamide ligands bind to human carbonic anhydrase with a conserved binding geometry, an enthalpy-driven binding, and indistinguishable binding affinities (see picture). These data support the pervasive theory that the lock-and-key model disregards an important component of binding: the water, which fills the binding pocket of the protein and surrounds the ligand.

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 and (ii) the delivery and expression of the complementing gene that turns on enzymatic activity and produces a colored or fluorescent product. Here we demonstrate a phage-based amplification scheme with an M13KE phage that delivers a small peptide motif to an F(+), α-complementing strain of Escherichia coli K12, which expresses the ω-domain of β-galactosidase (β-gal). The result of this complementation-an active form of β-gal-was detected colorimetrically, and the high level of expression of the ω-domain of β-gal in the model K12 strains allowed us to detect, on average, five colony-forming units (CFUs) of this strain in 1 L of water with an overnight culture-based assay. We also detected 50 CFUs of the model K12 strain in 1 L of water (or 10 mL of orange juice, or 10 mL of skim milk) in less than 4 h 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 potentially be extended to detect many different bacteria with bacteriophages that deliver genes encoding a full-length enzyme that is not natively expressed in the target bacteria.

Rapid determination of RNA accessible sites by surface plasmon resonance detection of hybridization to DNA arrays

RNA accessible sites are the regions in an RNA molecule that are available for hybridization with cDNA or RNA molecules. The identification of these accessible sites is a critical first step in identifying antisense-mediated gene suppression sites, as well as in a variety of other RNA-based analysis methods. Here, we present a rapid, hybridization-based, label-free method of identifying RNA accessible sites with surface plasmon resonance imaging (SPRi) on in situ synthesized oligonucleotide arrays prepared on carbon-on-metal substrates. The accessible sites of three pre-miRNAs, miRNA precursors of approximately 75 nt in length, were determined by hybridizing the RNA molecules to RNA-specific tiling arrays. An array composed of all possible 6mer oligonucleotide sequences was also utilized in this work, offering a universal platform capable of studying RNA molecules in a high throughput manner.

In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired.