Smart “lanthano” proteins for phospholipid sensing

Metal-ion-mediated interactions between calcium-binding peripheral proteins and membrane phospholipids is a key feature of multiple cell signaling processes. The molecular basis for the interaction involves the displacement of inner-sphere water molecules on calcium ions by phosphate groups of the phospholipids. On the basis of this fundamental mechanism, we have devised a novel “turn-on” optical sensing strategy for anionic phospholipids by using a lanthanide reconstituted protein. The “lanthano” protein turns on selectively in the presence of a crucial signaling phospholipid, phosphatidylserine, by affording a 6 times enhancement in lanthanide luminescence. The “turn-on” sensing strategy was distinctly validated by direct evidence for the water-displacement mechanism via lifetime measurements

A Bishydrated, Eight-Coordinate Gd(III) Complex with Very Fast Water Exchange: Synthesis, Characterization, and Phantom MR Imaging

International audienceWe report here the physical-chemical characterization of a highly water-soluble Gd(III) complex (complex 1) formed with the asymmetric hexadentate ligand H(4)bedik (2,2-((2-(bis (carboxymethyl)amino)benzyl)azanediyl)diacetic acid). The number of water molecules directly coordinated to the metal, q = 2.2, was assessed by measuring luminescence lifetimes of the Tb(III) analogue (complex 2) in H2O and D2O and it further was confirmed by O-17 chemical shift measurements on the Gd(III) complex. The complex has moderate thermodynamic stability and remains insensitive to physiological anions [biphosphate (HPO42-), bicarbonate (HCO3-)] and pH variation (in the range 5-10) of the medium as evidenced by negligible changes in longitudinal relaxivity (r(1)) at 1.41 T, 25 degrees C. The Gd(III) complex exhibits very fast water exchange, among the fastest reported for Gd(III) chelates, and high relaxivity at high magnetic fields (r(1) = 7.47mM(-1)s(-1) at 9.4 T, pH similar to 7 and 25 degrees C). Interestingly, the very high positive value of the activation entropy indicates a dissociatively activated water exchange for this eight-coordinate complex

Smart “Lanthano” Proteins for Phospholipid Sensing

Metal-ion-mediated interactions between calcium-binding peripheral proteins and membrane phospholipids is a key feature of multiple cell signaling processes. The molecular basis for the interaction involves the displacement of inner-sphere water molecules on calcium ions by phosphate groups of the phospholipids. On the basis of this fundamental mechanism, we have devised a novel “turn-on” optical sensing strategy for anionic phospholipids by using a lanthanide reconstituted protein. The “lanthano” protein turns on selectively in the presence of a crucial signaling phospholipid, phosphatidylserine, by affording a 6 times enhancement in lanthanide luminescence. The “turn-on” sensing strategy was distinctly validated by direct evidence for the water-displacement mechanism via lifetime measurements

Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides

Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI­(4,5)­P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI­(4,5)­P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling