Surface Plasmon Resonance Imaging of Transcription Factor Proteins:  Interactions of Bacterial Response Regulators with DNA Arrays on Gold Films

Abstract

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.

Bacterial 2-Component Signal Transduction Systems: A Fluorescence Polarization Screen for Response Regulator-Protein Binding

“…It is also comparable to the value of 40 nM reported for the binding of the entire vanHAX promoter region to VanR~P in a previously reported gel shift measurement 14 and to the value of approximately 150 nM reported for the binding of GST-VanR~P to each of the 3 promoter segments in the previously reported SPR study. 15 Compound A at 1.5, 2.5, and 5.0 µM increased the apparent K d to 35 nM, 150 nM, and 500 nM. This compares with the 4-to 5-fold increase reported for the apparent K d for the GSTVanR~P-promoter segment interactions reported in the SPR study.…”

Recent advances in surface plasmon resonance imaging: detection speed, sensitivity, and portability

“…So far, the intensity interrogation mode is a straightforward case for 2D arrays. It has been widely used and commercialized by Biacore GE and GWC Technologies for high-throughput analysis in immunosensing [4], DNA hybridization [7], protein interactions [9][10][11], and cellular analysis [12,13]. However, for the detection of multiple interactions simultaneously, it is usually not practical to obtain a homogeneous and optimal response over the entire analyzed surface with the choice of a unique operating point [14].…”

Nanoscale virus biosensors: state of the art

“…The prepared biosensor exhibited good sensitivity with a detection limit of 0.5 pM and was able to discriminate perfect complementary and possible mismatches in sequence. 216 Not only NAs, but also proteins, transcription factors [217][218][219] , and drugs 220 were determined using SPR via their interaction with NA. Fisher et al 221 tested recombinant HIV-1 nucleocapsid protein as a target to very short biotinylated oligonucleotides.…”