Combinational Application of Surface Plasmon Resonance Spectroscopy and Quartz Crystal Microbalance for Studying Nuclear Hormone Receptor-Response Element Interactions

摘要

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 ER(alpha) and ER(beta), (2) ER(alpha) has a higher sequence specificity than ER(beta) or there were more nonspecific interactions between ER(beta) and control DNA, and (3) ER(alpha) bound ERE as dimers and ER(beta) 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 (96percent 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 ER(alpha)-DNA and ER(beta)-DNA complexes.