Ligand binding by estrogen receptor beta attached to nanospheres measured by fluorescence correlation spectroscopy

Abstract

Although many indirect methods have been chosen to study the system of estrogen receptor ligand binding, an ideal method is fluorescence correlation spectroscopy (FCS). FCS is nondestructive to the sample, uses very small sample volumes, and operates well within physiological concentration ranges. The methodology was developed to biotinylate the estrogen receptor beta-ligand binding domain (ERbeta-LBD) using biotin with a very short spacer and to then attach this protein to a 40 nm neutravidin-coated bead (nanosphere). Diffusional FCS data were obtained for a fluorescently labeled coactivator peptide, steroid receptor coactivator peptide-1 (A-SRC-1(2)), in the absence and presence of bead-bound ERbeta-LBD. Data were also acquired in the presence of one of the endogenous ligands for ERbeta, 17beta-estradiol, and with tamoxifen. The bead strategy resulted in a decreased receptor diffusion coefficient and consequent increase in the decay time of the FCS autocorrelation functions for receptor-bound, labeled SRC-1(2). Thus, free and bound coactivators were much more readily distinguished by FCS. Discrimination between the fluorescently labeled unbound and bound species could be determined in autocorrelation functions obtained in as few as 30 s. The advantage of using FCS with the ERbeta-LBD: bead methodology is the ability to obtain reliable and reproducible data in a short time frame.