Surface plasmon resonance (SPR) imaging measurements have been used to study the sequence specific adsorption of response regulator proteins to DNA arrays constructed on gold thin films. Bacteria adapt to their environment primarily through two-component signal transduction systems that consist of an environmental sensor histidine kinase and a transcription activator response regulator DNA binding protein. DNA arrays were fabricated from oligonucleotide sequences of known DNA binding regions for two response regulators: OmpR, which controls gene expression of the outer membrane porin proteins in Escherichia coli, and VanR, which is involved in the antibiotic vancomycin resistance in Enterococcus faecium. The label-free method of SPR imaging was then used to monitor the sequence specific binding of these two response regulator proteins to the DNA arrays. The promoter regions from the ompF (F1 and F2) and ompC (C1) genes for the OmpR protein, and from the vanRS (R1) and vanHAX (H1 and H2) genes for the VanR protein were studied. SPR imaging was used to (i) monitor and compare the binding of both response regulators to various promoter regions on the DNA array, (ii) compare the binding of the OmpR response regulator protein in its phosphorylated and nonphosphorylated forms, and (iii) monitor the inhibition of VanR protein binding to the DNA arrays in the presence of a small molecule DNA binding inhibitor. The proteins exhibited specificity for the known binding sequences compared to control sequences on the DNA array. For the OmpR protein, the highest amount of binding was observed at the F1 site. When OmpR was phosphorylated by a small molecule phosphodonor, acetyl phosphate, there was on average a 42% increase in protein binding. In contrast, phosphorylated VanR binding decreased by an average of 40% in the presence of a known DNA binding inhibitor, (2,3,4-trifluorophenylisothiazolone). These measurements demonstrate that SPR imaging is an effective screening method for compounds that target DNA protein interactions and can serve as a useful tool for the discovery of new therapeutic molecules that target DNA binding proteins. † Part of the Langmuir special issue entitled The Biomolecular Interface.

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Purpose: To present a method for significantly increasing the concentration of a hyperpolarized compound produced by a commercial dynamic nuclear polarization (DNP) polarizer, enabling the polarization process to be more suitable for preclinical applications. Materials and Methods:Using a HyperSense DNP polarizer, we investigated the combined use of perfluorocarbon and water to warm and dissolve the hyperpolarized material from the polarization temperature of 1.4K to produce material at temperatures suitable for injection.Results: By replacing 75% of the water in the dissolution volume with a chemically and biologically inert liquid that is immiscible with water, the injection volume can be reduced 4-fold. Rapid separation of the water and perfluorocarbon mixture enables the aqueous layer containing polarized material to be easily and rapidly collected. Conclusion:The approach provides a significantly increased concentration of compound in a volume for injection that is more appropriate for small animal studies. This is demonstrated for 13 C-labeled pyruvic acid and 13 C-labeled succinate, but may be applied to the majority of nuclei and compounds hyperpolarized by the DNP method. THERE IS INCREASING INTEREST in using dynamic nuclear polarization (DNP) to enhance the available NMR signal from a variety of compounds and nuclei for use in magnetic resonance imaging (MRI) and spectroscopy studies. When using a commercial polarizer system (HyperSense, Oxford Instruments, Oxford, UK), the standard dissolution volume (output volume) is typically around 4ml and therefore not optimal for small animal research. The relatively large volume of water contains ethylenediaminetetraacetic (EDTA), along with a buffering agent, which is used to warm and extract a concentrated smaller volume (typically around 30 mL) of a frozen solution comprised of the polarized compound mixed with radical. Following this dissolution process, the resultant volume may be significantly more dilute than desired, or too large a volume for safe, rapid bolus injection into a small animal. The recommended injection volume and rate are 5 mL/kg and 0.05 mL/s for both mice and rats (1). For a 200 g rat this results in a typical injection of 1 mL taking 20 seconds. For a 20 g mouse, this becomes 0.1 mL in 2 seconds, which is a short, tight bolus, but it is a very small volume compared to the typical polarizer output. When following the recommended injection procedure, this results in only about 25% of the total polarized solution injected into an adult rat and only 2.5% of the solution for a mouse. The outcome is a significant waste of the hyperpolarized compound and an inordinately large bolus. In both cases this can result in a suboptimal study due to the large and dilute dissolution solution.Maximizing the signal available from 13 C-labeled compounds is critical for in vivo or in vitro imaging or spectroscopy. Consequently, this work explored the possibility of reducing the volume of injectable solution produced by the polarizer. To achieve this, we inv...

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Two-component signal transduction systems are the primary means by which bacteria sense environmental change and integrate an adaptive response. In pathogenic bacteria, 2-component signal transduction (TCST) kinases are involved in the expression of virulence and antibiotic resistance. This makes bacterial TCST systems attractive targets for pharmacologic intervention. This paper describes a fluorescence polarization assay that quantifies the binding between bacterial DNA promoter segments and their cognate response regulator proteins. Using the VanRSTCST system from Enterococcus faecium, which encodes vancomycin resistance, the authors demonstrate inhibition of response regulator protein/promoter segment binding with a known inhibitor. Observed binding constants were comparable to those reported in surface plasmon resonance measurements and gel shift measurements. (Journal of Biomolecular Screening 2005:270-274)