Specht AlexandreLaboratoire de Conception et Application de Molecules Bioactives, UMR 7199, CNRS/UDS, Faculte de Pharmacie, 74 Route du Rhin, 67400 Illkirch FrancePhone : Fax :
An uncaging process refers to a fast and efficient release of a biomolecule after photochemical excitation from a photoactivatable precursor. Two-photon excitation produces excited states identical to standard UV excitation while overcoming major limitations when dealing with biological materials, like spatial resolution, tissue penetration and toxicity and has therefore been applied to the uncaging of different biological effectors. A literature survey of two-photon uncaging of biomolecules is described in this article, including applications in cellular- and neurobiology.
Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime. The 'caged' arsenocholine was shown to bind at both the active and peripheral sites of acetylcholinesterase. UV irradiation of a complex with acetylcholinesterase during a brief temperature excursion from 100 K to room temperature is most likely to have resulted in a decrease in occupancy by the caged compound. Microspectrophotometric experiments showed that the caged compound had indeed been photocleaved. It is proposed that a fraction of the arsenocholine molecules released within the crystal had been expelled from both the active and the peripheral sites. Partial q-weighted difference refinement revealed a relative movement of the two domains in acetylcholinesterase after photolysis and the room-temperature excursion, resulting in an increase in the active-site gorge volume of 30% and 35% in monomers A and B of the asymmetric unit, respectively. Moreover, an alternative route to the active-site gorge of the enzyme appeared to open. This structural characterization of acetylcholinesterase 'at work' is consistent with the idea that choline exits from the enzyme after catalysis either via the gorge or via an alternative 'backdoor' trajectory.
Title: Photoreversible inhibitions of cholinesterases Loudwig S, Specht A, Goeldner M Ref: In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects, (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina:277 , 2004 : PubMed
Title: Poster (52) Photo-reversible inhibitions of cholinesterases Loudwig S, Specht A, Goeldner M Ref: In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects, (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina:348, 2004 : PubMed
Title: Mutually induced formation of host-guest complexes between p-sulfonated calix[8]arene and photolabile cholinergic ligands Specht A, Bernard P, Goeldner M, Peng L Ref: Angew Chem Int Ed Engl, 41:4706, 2002 : PubMed
