The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.

The 2.1-Å resolution crystal structure of wild-type green f luorescent protein and comparison of it with the recently determined structure of the Ser-65 3 Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the f luorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the f luorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the f luorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the f luorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore's excited state to Glu-222. Loss of the Ser-205-Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the f luorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green f luorescent protein should enable engineering of environmentally sensitive f luorescent indicators and UV-triggered f luorescent markers of protein diffusion and trafficking in living cells.The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is the first known protein in which visible fluorescence is genetically encodable. The fluorophore is derived from natural residues present within the primary structure of GFP, so no exogenous cofactor or substrate is needed for fluorescence (1, 2). The tremendous potential of GFP as a reporter of gene expression, cell lineage, and protein trafficking and interactions has been extensively reviewed (3-5).Wild-type (WT) GFP is a 238-aa protein (2). In vitro GFP is a particularly stable protease-resistant protein (6) and is only denatured under extreme conditions (7). The GFP chromophore, p-hydroxybenzylideneimidazolinone (8, 9), is formed by internal cyclization of a Ser-Tyr-Gly tripeptide and 1,2-dehydrogenation of the Tyr. This posttranslational modification is oxygendependent, requiring Ϸ2-4 h for the WT protein (10, 11). A mechanism for the fluorophore formation has been proposed (3) but needs to be confirmed by further studies.GFP absorbs blue light at 395 nm, with a smaller peak at 475 nm, and emits green light at 508 nm with a quantum yie...

Glycogen synthase kinase 3 (GSK3) is a serine/threonine kinase that has been implicated in pathological conditions such as diabetes and Alzheimer's disease. We report the characterization of a GSK3 inhibitor, AR-A014418, which inhibits GSK3 (IC 50 ‫؍‬ 104 ؎ 27 nM), in an ATP-competitive manner (K i ‫؍‬ 38 nM). AR-A014418 does not significantly inhibit cdk2 or cdk5 (IC 50 > 100 M) or 26 other kinases demonstrating high specificity for GSK3. We report the co-crystallization of AR-A014418 with the GSK3␤ protein and provide a description of the interactions within the ATP pocket, as well as an understanding of the structural basis for the selectivity of AR-A014418. AR-A014418 inhibits tau phosphorylation at a GSK3-specific site (Ser-396) in cells stably expressing human four-repeat tau protein. AR-A014418 protects N2A neuroblastoma cells against cell death mediated by inhibition of the phosphatidylinositol 3-kinase/protein kinase B survival pathway. Furthermore, AR-A014418 inhibits neurodegeneration mediated by ␤-amyloid peptide in hippocampal slices. AR-A014418 may thus have important applications as a tool to elucidate the role of GSK3 in cellular signaling and possibly in Alzheimer's disease. AR-A014418 is the first compound of a family of specific inhibitors of GSK3 that does not significantly inhibit closely related kinases such as cdk2 or cdk5.

Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimer's disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and blood-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimer's disease.

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