Characterization of Single Molecule Protein Induced Fluorescence Enhancement (PIFE) as an Alternative to SmFRET

Abstract

Forster resonance energy transfer (FRET) is a widely used technique for real time single molecule detection. FRET measurement is based on distance changes between donor and acceptor fluorophores, hence the requirement for fluorophore attachment of the molecules under study. Single fluorophore labeling of protein is often difficult to achieve, rendering poor efficiency and specificity of labeling. Furthermore, proteins with high Kd which requires addition of high protein concentration would make single molecule detection impossible even with properly labeled proteins. Recently, PIFE, an alternative fluorescence assay was developed for probing translocational movement of an antiviral protein, RIG-I (1). PIFE employs a single fluorophore which exhibits enhanced quantum yield when approached by a protein at a close proximity. Although this photophysical effect is correlated with the lifetime change of the corresponding fluorophore (2) the sensitivity and the distance range of the method needs to be further characterized.We performed a systematic study of a single molecule PIFE where we monitored binding of a restriction enzyme BamHI to Cy3 labeled DNA. Preliminary data shows that the sequence specific binding of BamHI to DNA in a buffer containing calcium results in an increase of Cy3 intensity. The overall level of fluorescence increase shows dependence on the protein concentration and single molecule traces display binding and unbinding as discrete steps of increase and decrease in Cy3 intensity respectively. We confirmed the sequence specific cleavage activity of BamHI triggered by the presence of magnesium.