Biophysical aspects of ligand-receptor interactions

Fluorescence correlation spectroscopy (FCS) has been used to obtain equilibrium dissociation data for the biotinylated estrogen receptor β ligand binding domain (B-ERβ-LBD) attached to a 40 nm neutravidin coated bead in the presence of the Alexa488 labeled steroid receptor coactivator-1 (2) (A-SRC-1(2)) and the ligand, 17β-estradiol. These data yielded a K_d of 0.22 nM ± 0.06 nM. Previous studies using estrogen receptor α ligand binding domain (ERα-LBD) and SRC-1(2) have experimentally determined a K_d of 30 nM ± 6 nM. Additional equilibrium data have been obtained for the interaction of A-SRC-1 (2) and B-ERβ-LBD both free in solution as well as attached to 40 nm neutravidin coated beads. The strategy of attaching the B-ERβ-LBD to a neutravidin coated bead was to increase the decay time associated with the bound A-SRC-1(2) thereby increasing the accuracy with which the equilibrium dissociation constant can be determined. Numerous calibration measurements were done using Alexa labeled ERβ-LBD (A-ERβ-LBD) to verify experimental and biochemical conditions.; Fluorescence pattern photobleaching recovery (FPPR) using a Ronchi ruling, fluorescence photobleaching recovery (FPR) using a focused spot, and total internal reflection fluorescence photobleaching recovery (TIR-FPR) using evanescent excitation are all techniques used in the Thompson lab. To control the optical shutters, the photomultiplier (PMT) shutter, and to monitor counts from the PMT, LabVIEW software was used to write two virtual instruments (VI) that control and monitor the timing and function of the equipment and computer. The “CheckSample.VI” is responsible for checking the sample intensity to verify it is within acceptable experimental parameters, and the “NewAlgorithm1.VI” is responsible for computer and equipment function timing, and data collection during an actual experiment. This software has been tested under various controlled conditions, and with actual experimental samples. Testing, analysis, documentation, annotation and schematics are presented in this dissertation.; The general solution to the fundamental rebinding probability of a ligand rebinding to a sphere has been presented. The theory for a ligand rebinding at planar surfaces has been previously derived. This mathematical treatment approaches the conditions of the ligand rebinding to a spherical surface, which is more applicable to ligand-receptor interactions on biological cell membranes.