This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Abstract Dr. Andrew Bradbury of the Bioscience Division is developing a new class of fluorescent ligand termed 'fluorobodies', which are GFP molecules to which a series of ligand-recognition loops have been added at one end, producing a quasi-antibody binding site (1). Such fluorobodies would be much more useful if there was some change in the fluorescence signal upon binding. We will investigate whether binding of fluorbodies to their target ligand induces a measurable change in the fluorescence lifetime of the GFP. If the bound and unbound molecules had two different fluorescence lifetimes, this would essentially be a switch that detected binding. We will measure the fluorescence lifetime of fluorobodies bound to microspheres exposed to differing concentrations of ligand, and determine if we can detect a difference in shift in lifetime upon binding. Dr. Bradbury's group has engineered several different GFP molecules that vary in the robustness of their folding, and we may be able to select larger lifetime effects of ligand binding by using GFPs having less stable protein folding. Background We are developing a new class of fluorescent ligand termed 'fluorobodies'. These are GFP molecules into which antibody binding loops have been inserted. In its present incarnation, single loops corresponding to the third hypervariable region of the heavy chain variable region have been inserted into a specific site in GFP. A library of such fluorobodies has been created using random HCDR3's derived from lymphocytes (see attached publication in appendix) and specific binders selected by phage display. Even though only single loops are displayed within the GFP, he has been able to select binders recognizing a number of different targets with affinities in the high nanomolar range (400-1000nM). This is significantly higher than the affinities obtained by traditional peptide phage display, and the selected binders retain their fluorescence. In fact, the determination of the affinity was carried out using flow cytometry with antigen coupled to polystyrene beads and the detected fluorescence arising from fluorobody binding. Approach Fluorobodies would be much more useful if there was some change in the fluorescence signal upon binding. Although we are attempting to develop binders based on fluorescent proteins that change their fluorescence upon binding, an alternative approach is to determine whether other fluorescence properties change upon binding. We will investigate whether binding of fluorobodies to their target ligands induces a measurable change in the fluorescence lifetime of the GFP. If the bound and unbound molecules had two different fluorescence lifetimes, this would essentially be a switch that detected binding. We will take an evolving approach to the implementation and application of this technology. First, we will measure the fluorescence lifetimes of fluorobodies bound to microspheres and exposed to differing concentrations of ligand, and determine if we can detect a difference in shift in lifetime upon binding. This work can be done with the upgraded version of the separated PS cytometer. Second, we will determine if we can engineer fluorobodies with enhance lifetime changes upon binding. We have already engineered several different fluorescent proteins that vary in the robustness of their folding, and we may be able to select larger lifetime effects of ligand binding by using GFPs having less stable protein folding. This application will take good advantage of the capability of measuring multiple lifetimes simultaneously, to be implemented on both the phase sensitive and integrated phase-spectral instruments. Third, we will determine whether we can develop fluorobodies with different spectral emission spectra, e.g. YFP, BFP. This would then open the potential to use combinations of fluorobodies with differing emission spectra to simultaneously to quantitate bound and unbound forms of several ligands in cells and bead-based assays. If this technology is successful, it will provide a potentially very powerful biological application for the integrated phase-spectral instrument, which would provide the ability to distinguish spectral emission and lifetime on a mixture of many fluorobodies.