Na ؉ -H ؉ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodiumphosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser 290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1␣ (PP1␣), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser 290 . Mutating 257 VPF 259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1␣-mediated Ser 290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ␤-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally . 4 The abbreviations used are: PTH, parathyroid hormone; PTHR, PTH receptor; NHERF1, Na ϩ /H ϩ -exchanger regulatory factor 1; GnTI Ϫ , N-acetylglucosaminyltransferase-deficient HEK-293S cells; TAP-NHERF1, tandem affinity purification-tagged NHERF1; FLIM, fluorescence lifetime imaging; HDX, hydrogen/deuterium exchange mass spectrometry; pSer 290 , phosphorylated Ser 290 ; NP40, Nonidet P-40; TAMRA, carboxytetramethylrhodamine; SILAC, stable isotope labeling of amino acids in cell culture; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; ND, nondeuterated; FD, fully deuterated; EBD, ezrin-binding domain; PDB, Protein Data Bank; 2Me-4OMe-TM, 7-hydroxy-5,5-dimethyl-10-(4-methoxy-2-methylphenyl)-dibenzo-[b,e]-silin-3(5H)-one; ANOVA, analysis of variance; ID, intrinsically disordered; TTN, tautomycetin; RPTEC, renal proximal tubule epithelial cell; pen/strep, penicillin and streptomycin; CFP, cyan fluorescent protein; FERM, Ezrin-Radixin-Moesin; MD, molecular dynamics.

2',7'-Difluorofluorescein (Oregon Green 488, OG488) is a novel fluorescein dye derivative which presents important advantages for improving the fluorimetric applications in the biomedical and biochemical sciences. In aqueous solution it displays four prototropic forms, namely cation (C), neutral (N), monoanion (M), and dianion (D). In previous works, we found (J. Phys. Chem. A 2005, 109, 734-747, 2840-2846) that OG488 undergoes excited-state proton transfer reactions, which may affect the results from applications using this dye. We established that the excited-state proton transfer (ESPT) reactions between neutral, monoanionic, and dianionic forms of OG488 are promoted by acetate buffer, and we characterized the ground and excited species involved. We also solved the kinetics of the prototropic reactions using global compartmental analysis. In the present paper, we extend our study on the ESPT reactions of OG488 to acidic media, in which only the three prototropic species cation, neutral, and monoanion coexist. We have solved the kinetics of the three-state ESPT reaction by means of global three-compartmental analysis of a fluorescence decay surface in moderately acidic media (pH between 1.1 and 3.0), recovering the kinetic and spectral parameters of this three-state system. This system is one of the most complex solved to date, due to the strong overlap of the absorption and emission spectra of the neutral and monoanionic forms of OG488. We also found that the cation behaves as "super" photoacid, showing a very high deprotonation rate constant (1.04 x 10(11) s(-1)) and an enhanced acidity. Therefore, we also carried out experiments at very high perchloric acid concentrations, dealing with some other effects which become noteworthy at these [H(+)]. The presence of xanthylium cation quenching due to "free" water molecules, and the reduction in the amount of water clusters acting as proton acceptors, are processes which alter notably the time course of the excited-species in this high [H(+)] range.

Herein, we describe the synthesis and application of cross-linked polystyrene-based dual-function nano- and microparticles containing both fluorescent tags and metals. Despite containing a single dye, these particles exhibit a characteristic dual-band fluorescence emission. Moreover, these particles can be combined with different metal ions to obtain hybrid metallofluorescent particles. We demonstrate that these particles are easily nanofected into living cells, allowing them to be used for effective fingerprinting in multimodal fluorescence-based and mass spectrometry-based flow cytometry experiments. Likewise, the in situ reductions of the metal ions enable other potential uses of the particles as heterogeneous catalysts.

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