Luminescent probes for the detection of glutamine and protein hydrodynamics

Abstract

We have prepared novel fluorescent probes for protein hydrodynamics and for the determination of glutamine in solution. Ru(II) and Re(I) metal ligand complexes (MLCs) have been synthesized with amine- and sulfhydryl-reactive moieties suitable for modifying biomolecules. The spectral properties of MLCs when free in solution and when conjugated to proteins have been studied. We have characterized MLCs which display photoluminescent lifetimes between 60 ns and 3.5 mus and possess useful anisotropy values between 0.1 and 0.32. Time resolved anisotropy measurements were used to demonstrate the potential utility of these complexes in measuring the long rotational correlation times of macromolecules in solution. The usefulness of this class of molecules in fluorescence polarization immunoassay (FPI) was explored using an association reaction between HSA and polyclonal anitbody. A series of water soluble MLCs have been synthesized and characterized which may be used to measure correlation times between 0.1 and 10 mus. We have developed a reagentless optical assay for glutamine based on the Escherichia coli glutamine binding protein (GlnBP). Site-directed mutagenesis was performed to engineer single cysteine mutants which were covalently modified with environmentally sensitive sulfhydryl-reactive probes. The fluorescence emission of acrylodan and 2-(4'(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS) attached to GlnBP mutant S179C was shown to decrease 65 and 35 percent, respectively, upon titration with increasing amounts of glutamine (0 to 6.4 muM; KDapp 160 nM). No significant changes in the fluorescence intensity were observed for the structurally similar amino acids glutamate, asparagine and arginine. Time-resolved intensity decays showed a 2.4-fold decrease in mean lifetime for GlnBP S179C-acrylodan upon the addition of glutamine, indicating the possibility of a lifetime-based assay. Anisotropy decay measurements for GlnBP S179C-acrylodan showed a 13 ns rotational correlation time in the ligand free state; whereas, multiple correlation times were assigned in the glutamine bound conformation. The decrease in fluorescence intensity of S179C-acrylodan was adapted to polarization sensing of glutamine. A generic approach to the design of ratiometric biosensors is also discussed. The engineered GlnBP is a first step towards the development of a nonenzymatic biosensor capable of determining glutamine concentrations in cell cultures.