Characterization of protein--DNA interactions using surface plasmon resonance spectroscopy with various assay schemes.
Specific protein-DNA interactions play a central role in transcription and other biological processes. A comprehensive characterization of protein-DNA interactions should include information about binding affinity, kinetics, sequence specificity, and binding stoichiometry. In this study, we have used surface plasmon resonance spectroscopy (SPR) to study the interactions between human estrogen receptors (ER, alpha and beta subtypes) and estrogen response elements (ERE), with four assay schemes. First, we determined the sequence-dependent receptors' binding capacity by monitoring the binding of ER to various ERE sequences immobilized on a sensor surface (assay format denoted as the direct assay). Second, we screened the relative affinity of ER for various ERE sequences using a competition assay, in which the receptors bind to an ERE-immobilized surface in the presence of competitor ERE sequences. Third, we monitored the assembly of ER-ERE complexes on a SPR surface and thereafter the removal and/or dissociation of the ER (assay scheme denoted as the dissociation assay) to determine the binding stoichiometry. Last, a sandwich assay (ER binding to ERE followed by anti-ER recognition of a specific ER subtype) was performed in an effort to understand how ERalpha and ERbeta may associate and compete when binding to the DNA. With these assay schemes, we reaffirmed that (1) ERalpha is more sensitive than ERbeta to base pair change(s) in the consensus ERE, (2) ERalpha and ERbeta form a heterodimer when they bind to the consensus ERE, and (3) the binding stoichiometry of both ERalpha- and ERbeta-ERE complexes is dependent on salt concentration. With this study, we demonstrate the versatility of the SPR analysis. With the involvement of various assay arrangements, the SPR analysis can be further extended to more than kinetics and affinity study.
Teh
Huey Fang
HF
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602, and Genome Institute of Singapore, 60 Biopolis Street, Singapore 117528.
Peh
Wendy Y X
WY
Su
Xiaodi
X
Thomsen
Jane S
JS
D000818:Animals; D001483:Base Sequence; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D047628:Estrogen Receptor alpha / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D020218:Response Elements; D013057:Spectrum Analysis; D020349:Surface Plasmon Resonance
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D011994:Recombinant Proteins; D004247:DNA