Synthetic and Genetically Encoded Fluorescence Probes for Quantitative Analysis of Protein Hydrodynamics

Abstract

The fluorescence polarization technique that Prof. Weber developed at Cambridge University between the late 1940s and early 1950s has had a tremen- dous impact on our understanding of the structure and dynamics of macromolecules and in the analysis of proteins interactions and detection of target proteins in biologically complex samples. His decision to develop dimethylaminonaphthalene sulfonyl chloride (Dansyl-Cl) as the first probe for fluorescence polarization studies was brilliant, as its long fluorescence lifetime and well-defined dipole are ideally suited to study protein conjugates as large as 100 kDa. Indeed, after almost 70 years, the Dansyl group is still the probe of choice for in vitro applications of fluorescence polarization. Unfortunately, Dansyl is not very suitable for related studies in living cells, primarily because it requires excitation in the near ultraviolet, while the in vivo labeling of a target protein with Dansyl group is challenging. We have developed a new class of genetically encoded fluorescent protein thatmay help to overcome these limitations. The lumazine-binding protein (LUMP) harbors a fluorescent probewith a cerulean-colored emission that like Dansyl has a long excited state lifetime (14 ns). Moreover, LUMP has a smaller mass than GFP that allows us to genetically append capture sequences as large as 20 kDa and still generate a fusion protein with sufficient dynamic range in the fluorescence polarization value to quantify the amounts of the free and target-bound states in an equilibrium. In this article, I will compare and contrast key features of Dansyl and LUMP as probes for fluorescence polarization studies and discuss the potential of using LUMP and related encoded proteins to advance the application of fluorescence polarization to analyze target proteins and protein interactions in living cells. G.