Development of an oil/water TIRF-RET instrument for the study of interfacial protein adsorption, packing, and conformational change

Protein adsorption at interfaces is an important phenomenon with significant applications in fields such as pharmaceuticals and the chemical industry. Most work in this field has focused on adsorption behavior. Very little effort has been directed towards the study of protein packing or conformational changes associated with the adsorption. Here we develop a reflection spectrometer to study these phenomena and examine the interplay between them.; The work builds on a previously developed total internal reflection fluorescence spectrometer (TIRFS), and adds the technique of resonance energy transfer (RET). RET permits determination of the distance between two fluorescent moieties. Appropriate attachment of fluorescent dyes allows the TIRF-RET instrument to study either protein packing or conformational change in conjugation with the adsorption dynamics.; Classical electrodynamics calculations examined the validity of applying RET in the presence of an oil/water interface. Forster theory, which relates the efficiency of energy transfer and the distance between the fluorophores, was shown to hold to within +/-10% in the presence of an oil/water interface.; Intermolecular TIRF-RET experiments provide strong evidence against the possibility of associative adsorption of BSA at the oil/water interface. Long-term adsorption experiments showed that the protein adsorbed in a rapid exponential manner initially, followed by a much slower adsorption process, corresponding to a doubling of the adsorbed protein over the course of 48 hours. Such results have not been reported previously in the literature. Intramolecular TIRF-RET measurements were greatly complicated by the phenomena of fluorescence self-quenching. This experimental artifact made interpretation of data difficult at high protein surface-coverages. Furthermore, the slow mass transfer of the instrument limits its ability to probe short events, restricting the apparatus to a narrow window of time scales. Although we hypothesize that protein conformational change occurs at the interface, it has not been possible to obtain direct evidence with this instrument. Recommendations to solve the problem of fluorescence self-quenching, and for a redesigned instrument, are made in the last section.