Electric field-induced direct delivery of proteins by a nanofountain probe.

We report nanofabrication of protein dot and line patterns using a nanofountain atomic force microscopy probe (NFP). Biomolecules are continuously fed in solution through an integrated microfluidic system, and deposited directly onto a substrate. Deposition is controlled by application of an electric potential of appropriate sign and magnitude between the probe reservoir and substrate. Submicron dot and line molecular patterns were generated with resolution that depended on the magnitude of the applied voltage, dwell time, and writing speed. By using an energetic argument and a Kelvin condensation model, the quasi-equilibrium liquid-air interface at the probe tip was determined. The analysis revealed the origin of the need for electric fields in achieving protein transport to the substrate and confirmed experimental observations suggesting that pattern resolution is controlled by tip sharpness and not overall probe aperture. As such, the NFP combines the high-resolution of dip-pen nanolithography with the efficient continuous liquid feeding of micropipettes while allowing scalability to 1- and 2D probe arrays for high throughput.

Correlation of carboxylic acid pKa to protein binding and antibacterial activity of a novel class of bacterial translation inhibitors.

Previously we reported the discovery and initial optimization of a novel anthranilic acid derived class of antibacterial agents which suffered from extensive protein binding. This report describes efforts directed toward understanding the relationship of the acidity of the carboxylic acid with the extent of protein binding. The pK(a) of the acid was modified via the synthesis of a number of anthranilic acid analogs which vary the aromatic ring substituent at the 4-position. The pK(a) and HSA binding constants have been determined for each of the analogs. Our results indicate a correlation between pK(a) and HSA K(d). The physical properties and antibacterial activities will be discussed as well as how these results help address the protein binding issue with this series of compounds.

Multi-selective one dimensional proton NMR experiments for rapid screening and binding affinity measurements.

High-throughput ligand-based proton NMR screening performed in the presence of a spy molecule and a control molecule is a valuable tool for identifying drug leads. A limitation of the technique is represented by the severe overlap encountered in the screening of large chemical mixtures. An approach for overcoming this overlap problem is the use of multi-selective R(1) filtered and COSY or TOCSY experiments. Application of this methodology to compounds binding to the Sudlow site I of human serum albumin is presented. The screening is performed by simply monitoring the intensity of two signals. The precise measurement of the relative intensity of the two resonances permits determination of the binding constant of the NMR-hit. For a simple competition binding mechanism, the rapidly-derived NMR binding constants are in good agreement with the values derived from full-titration ITC and fluorescence spectroscopy measurements.

X-ray crystal structure of Staphylococcus aureus FemA.

The latter stages of peptidoglycan biosynthesis in Staphylococci involve the synthesis of a pentaglycine bridge on the epsilon amino group of the pentapeptide lysine side chain. Genetic and biochemical evidence suggest that sequential addition of these glycines is catalyzed by three homologous enzymes, FemX (FmhB), FemA, and FemB. The first protein structure from this family, Staphylococcus aureus FemA, has been solved at 2.1 A resolution by X-ray crystallography. The FemA structure reveals a unique organization of several known protein folds involved in peptide and tRNA binding. The surface of the protein also reveals an L-shaped channel suitable for a peptidoglycan substrate. Analysis of the structural features of this enzyme provides clues to the mechanism of action of S. aureus FemA.