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Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (P i ) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of P i binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni 2+ -affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the P i release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a P i detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Human amniotic fluid is of both maternal and fetal origin; it protects the fetus and provides the environment for growth and development of the fetus. We used a proteomics-based approach for targeting and purifying human phosphate binding protein, a member of the DING family of proteins from amniotic fluid, using Blue Sepharose CL-6B, DEAE-Sephacel and gel filtration chromatography. The protein had earlier been reported to be serendipitously purified along with PON1 (paraoxonase 1). It was identified using electro-spray-ionization-time-of-flight mass spectrometry and was found to be human phosphate binding protein. Human phosphate binding proteins have been reported to play a role as phosphate scavengers and may have a protective function against phosphate-related disorders, such as atherosclerosis, diabetes and kidney stones.

The Human Phosphate Binding Protein (HPBP) is a serendipitously discovered apolipoprotein from human plasma that binds phosphate. Amino acid sequence relates HPBP to an intriguing protein family that seems ubiquitous in eukaryotes. These proteins, named DING according to the sequence of their four conserved N-terminal residues, are systematically absent from eukaryotic genome databases. As a consequence, HPBP amino acids sequence had to be first assigned from the electronic density map. Then, an original approach combining X-ray crystallography and mass spectrometry provides the complete and a priori exact sequence of the 38-kDa HPBP. This first complete sequence of a eukaryotic DING protein will be helpful to study HPBP and the entire DING protein family., ((c) 2007 Wiley-Liss, Inc.)

Ras proteins are small GTPases that exhibit high-affinity binding to GDP and GTP and hydrolyze bound GTP to GDP. The intrinsic GTPase activity of Ras proteins is accelerated by GTPase activating proteins (GAPs), which act to attenuate GTPase signaling by accelerating the conversion of bound GTP to bound GDP. Tumor-associated Ras proteins harbor single amino acid substitutions at residues Gly-12 and Gln-61 that impair the intrinsic and GAP-stimulated GTPase activity, thus rendering these mutant Ras proteins persistently GTP bound and active in the absence of extracellular stimuli. The measurement of GTP hydrolysis in vitro can provide information on the intrinsic activity of, as well as help define, the GAP specificity. Current methods to measure GTP hydrolysis in vitro use either radioactivity-based filter binding assays or measurements of GDP:GTP:P(i) ratios by high-performance liquid chromatography (HPLC). Both provide only endpoint information on the GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here uses a fluorescently labeled, phosphate-binding protein (PBP) sensor. A change of protein conformation, caused by binding to a single P(i), is coupled to a measurable increase in fluorescence of the fluorophore. Therefore, this method does allow for real-time monitoring of GTPase activity. This chapter describes the preparation and labeling of the PBP with the MDCC fluorophore and its subsequent use in the measurement of GAP-stimulated GTPase activity. We have used the Ras family small GTPase R-Ras and the GAP-related domain from neurofibromin to demonstrate the application of these protocols.

To understand the physiology of non-differentiating bacteria exposed to nutrient deprivation and stress, various approaches have been employed in combination with detailed analysis of protein synthesis pattern. In this study, separation of proteins from clarified cell extracts of Pseudomonas aeruginosa PAO1 grown under phosphorus limiting conditions was achieved by high resolution two-dimensional gel electrophoresis (2-DE). Limitation of phosphate in the growth medium revealed significant differences in the 2-DE pattern of proteins between phosphate starved cells and an unstarved control. A major protein identified as PstS, a phosphate binding protein of the pts operon was exclusively found on 2-DE gels of phosphate starved bacteria. The identity of protein was established based on the results of Edman degradation, amino acid analysis and mass spectrometry. PstS was also found in other pseudomonads, and therefore, it can be used as a landmark protein in proteomic studies. Additionally, we propose utilizing pstS of pseudomonads for testing bioavailable phosphate from soils and water streams.