Detecting macromolecular associations by fluorescence spectroscopy

Fluorescence spectroscopy was the main biophysical technique chosen to examine the association of macromolecular complexes for two completely unique projects. In the first project, the selection and targeting of cytosolic ribosomes to the endoplasmic reticulum (ER) membrane was investigated by incorporating a fluorescent probe into signal sequences of ribosome•nascent chain complexes (RNCs). Having a fluorescent probe in the signal sequence allowed us to detect and quantify the interaction between the signal recognition particle (SRP) and RNCs. The resulting spectral data allowed us to determine, at equilibrium, the binding affinities of SRP for various signal sequence-containing RNCs (K_d = 0.05–0.38 nM). Competition experiments also showed that SRP can distinguish between translating and non-translating ribosomes because SRP binds to non-translating ribosomes (K_d = 71 nM) and to translating ribosomes that lack a signal sequence (K_d = 8 nM). By directly measuring the effect of GTP and GDP on SRP binding to RNCs, we found that SRP binding to signal sequences is unaffected by GTP or GDP. Therefore, we conclude that the process of ribosome selection for binding to the ER membrane involves a very high affinity interaction between the signal sequence of an RNC and SRP, and selection is accomplished without any involvement of GTP.; In the second project, fluorescence spectroscopy was used to detect perfringolysin O (PFO) binding to a membrane surface, oligomerizing, and ultimately forming a pore in the bilayer. PFO, a water-soluble bacterial cytolysin secreted by Clostridium perfringens, binds and forms large pores only in cholesterol-containing membranes, but the mechanism of the cholesterol-PFO interaction is still unknown. We therefore employed multiple different fluorescence techniques and were able show that the amount of membrane cholesterol required for PFO to interact with large unilamellar vesicles was mildly dependent on the length and degree of saturation of the phospholipid acyl chains. We also discovered that PFO binding to cholesterol-containing liposomes was not dependent on the measured membrane fluidity, nor on the presence of lipid rafts or lipid-ordered states. Instead, PFO binding to membranes appears to depend on the exposure of sufficient amounts of cholesterol to the toxin at the membrane surface.