1486 Langmuir 2003, 19, 1486-1492 Surface Plasmon Resonance Imaging of Transcription Factor Proteins: Interactions of Bacterial Response Regulators with DNA Arrays on Gold Films† Emily A. Smith,‡ Matthew G. Erickson,§ Andrew T. Ulijasz,| Bernard Weisblum,| and Robert M. Corn*,‡ Department of Chemistry, University of Wisconsin, 1101 University Avenue, Madison, Wisconsin 53706, Department of Medicine, University of Wisconsin, 600 Highland Avenue, Madison, Wisconsin 53792, and Pharmacology Department, University of Wisconsin, 1215 Linden Drive, Madison, Wisconsin 53706 Received February 6, 2002. In Final Form: April 19, 2002 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. Introduction to a collection of similar DNA sequences, which can exist in the promoter region of one or more genes. It is this The interactions between transcription regulatory sequence-specific binding of the transcription factors that proteins and their DNA binding sites are key aspects in underlies their role in the control of gene expression. the control of gene expression and the regulation of genetic Traditional methods that have been used to analyze DNA- information. The general structural motif that is found in protein interactions include gel shift analysis,5 DNA the DNA binding region of many transcription regulatory footprinting,6 and fluorescence polarization.7 These meth- - - proteins, the helix turn helix structure, can be altered ods provide reliable results but do not lend themselves to to recognize different target sequences through variations multiplexed studies of DNA-protein interactions. in the amino acid side chains or the linker that connects Surface plasmon resonance (SPR) imaging is a surface- 1-3 - the structural domains. Unlike DNA DNA hybridiza- sensitive spectroscopic technique that can be used to tion interactions responsible for the genetic code, the simultaneously monitor the interactions of many biomol- interactions between an amino acid and a nucleic acid ecules immobilized on a thin gold film without the use of 4 base pair are not a one to one relationship. Therefore, the fluorescent, enzymatic, or radioactive tags. SPR imaging DNA region that a particular transcription factor binds has been used previously to monitor DNA-DNA hybrid- to cannot necessarily be deduced from the protein’s amino ization, RNA-DNA hybridization, and DNA-protein acid sequence. In addition, many of these proteins bind interactions on surfaces.8-11 For example, we have recently - † Part of the Langmuir special issue entitled The Biomolecular (5) Sato, N.; Ohta, N. Nucleic Acids Res. 2001, 29, 2244 2250. (6) Tsung, K.; Brissette, R. E.; Inouye, M. J. Biol. Chem. 1989, 264, Interface. - ‡ 10104 10109. Department of Chemistry, University of Wisconsin. (7) Shoeman, R. L.; Hartig, R.; Traub, P. Biochemistry 1999, 38, § Department of Medicine, University of Wisconsin. 16802-16809. | Pharmacology Department, University of Wisconsin. (8) Smith, E. A.; Wanat, M. J.; Cheng, Y.; Barreira, S. V. P.; Frutos, (1) Mu¨ ller, C. W. Curr. Opin. Struct. Biol. 2001, 11,26-32. A. G.; Corn, R. M. Langmuir 2001, 17, 2502-2507. (2) Jen-Jacobson, L.; Engler, L. E.; Jacobson, L. A. Structure 2000, (9) Nelson, B. P.; Grimsrud, T. E.; Liles, M. R.; Goodman, R. M.; 8, 1015-1023. Corn, R. M. Anal. Chem. 2001, 73,1-7. (3) Doudna, J. A.; Richmond, T. J. Curr. Opin. Struct. Biol. 2001, 11, (10) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 11-13. 1999, 121, 8044-8051. (4) Stormo, G. D.; Fields, D. S. Trends Biochem. Sci. 1998, 23, 109- (11) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. 113. Chem. 2000, 51,41-63. 10.1021/la025609n CCC: $25.00 © 2003 American Chemical Society Published on Web 06/27/2002 Imaging of Transcription Factor Proteins Langmuir, Vol. 19, No. 5, 2003 1487 shown that SPR imaging experiments can be used to known DNA binding regions for two response regulator monitor the interactions of single-stranded binding protein proteins: OmpR, a well-studied response regulator that and the mismatch binding protein, MutS, to surface-bound controls the synthesis of the outer membrane porin DNA arrays.10,11 Studying sequence-specific DNA-protein proteins,15 and VanR, a response regulator involved in interactions in an array format with SPR imaging provides the antibiotic vancomycin resistance.17 SPR imaging was the advantage of examining the interactions of a particular then used to monitor the binding of OmpR and VanR protein with many DNA sequences (possibly the entire proteins to these DNA arrays. The amount of protein genome) in one step. DNA arrays are easily constructed binding to the various promoter segments was quantitated and can be designed to include sequences from the and compared. We also monitored and compared the promoter regions of one or many genes, or they can be binding of the response regulators to these DNA arrays constructed from a combinatorial mixture of nucleotides. in their phosphorylated (OmpR-P or VanR-P) and non- In addition, SPR imaging measurements with DNA arrays phosphorylated (OmpR or VanR) forms. Finally, we offer the possibility of directly studying proteins from a studied the binding of the VanR protein in the presence sample without the need for protein modification (i.e., of a known DNA binding inhibitor, CpdA, to show that fluorescent tagging) or prior knowledge of the protein’s this method is also feasible for screening potential DNA structure or function. binding inhibitors of response regulator proteins. This In this paper, we report the use of DNA arrays and SPR last measurement demonstrates that SPR imaging may imaging to study the sequence-specific binding of tran- be useful in the discovery of compounds that disrupt the scription regulatory proteins known as response regula- formation of response regulator-promoter DNA complexes tors. Response regulators are part of bacterial two- and alter gene expression in bacteria. component signal transduction systems, the primary means by which bacteria sense and adapt to environmental Experimental Considerations changes.12-14 These systems consist of two conserved components, a sensor histidine kinase protein that auto- Materials. Sulfosuccinimidyl 4-(N-maleimidomethyl) cyclo- hexane-1-carboxylate (SSMCC, Pierce), 11-mercaptoundecyl- phosphorylates in the presence of an appropriate envi- amine (MUAM, Dojindo Laboratories), 9-fluorenylmethoxycar- ronmental stimulus and a response regulator protein. Both bonyl-N-hydroxysuccinimide (FMOC-NHS, Novabiochem), N-hy- response regulator and sensor kinase proteins are phos- droxysuccinimidyl ester of methoxypoly(ethylene glycol) propionic phoproteins that can exist in an “active” (phosphorylated) acid MW 2000 (PEG-NHS, Shearwater Polymers), acetyl phos- and an “inactive” (unphosphorylated) form. A phospho- phate lithium potassium salt, HEPES, magnesium chloride, and rylated sensor kinase can convert a response regulator urea (Sigma), sodium chloride (Aldrich), disodium hydrogen from its inactive form to its active form, which has an phosphate dihydrate and sodium dihydrogen phosphate mono- increased affinity for a particular DNA binding site. In hydrate (Fluka), and 2,3,4-trifluorophenylisothiazolone (CpdA, bacteria, these two-component systems typically control Maybridge Chemical Co.) were used as received. All rinsing steps were performed with absolute ethanol and Millipore-filtered transcriptional activation of genes by binding upstream