Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptor-response element interactions.
Conventional methodologies for studying protein-DNA complexes, such as electrophoretic mobility shift assays (EMSAs), lack the real-time sensitivity and precision to accurately characterize the complex dynamics of interactions between transcription factors and their binding sites. To better understand the interactions between estrogen receptor (ER) subtypes and the estrogen response elements (EREs), we employed surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance with dissipation measurement (QCM-D) and made the following observations: (1) base substitutions in ERE half-sites reduced binding affinity for both ERalpha and ERbeta, (2) ERalpha has a higher sequence specificity than ERbeta or there were more nonspecific interactions between ERbeta and control DNA, and (3) ERalpha bound ERE as dimers and ERbeta bound as tetramers. These findings highlight intrinsic differences in DNA-binding properties between receptor subtypes, which are not apparent based on the high degree of conservation (96% identity) in their DNA-binding domains and results from EMSA studies. With this study, we demonstrate the potential of utilizing SPR and QCM in combination for a comprehensive characterization of ER-DNA interactions, including sequence-dependent binding mechanisms and structural differences in ERalpha-DNA and ERbeta-DNA complexes.
Su
Xiaodi
X
Institute of Materials Research and Engineering, 3 Research Link, Singapore 117602. xd-su@imre.a-star.edu.sg
Lin
Chin-Yo
CY
O'Shea
Sean J
SJ
Teh
Huey Fang
HF
Peh
Wendy Y X
WY
Thomsen
Jane S
JS
D004247:DNA / Q000032:analysis; D006801:Humans; D011189:Potassium Chloride / Q000737:chemistry; D011791:Quartz; D011960:Receptors, Estrogen / Q000032:analysis*; D020218:Response Elements; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D013997:Time Factors
D011960:Receptors, Estrogen; D011791:Quartz; D011189:Potassium Chloride; D004247:DNA