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 The advent of optogenetics provided researchers Alerter l'administrateur Recommander à un ami Lien de l'article 

The advent of optogenetics provided researchers with powerful tools that manipulate living systems with exquisite spatiotemporal precision using non-invasive visible light. The plethora of optical reporters to probe changes in cellular structure, second messenger levels, post-translational modifications and even conformation of individual proteins was until recently matched by few optical actuators, mostly chemically caged compounds released by ultraviolet light. Synthetic biology has now provided numerous designer channels and proteins that can be activated in different cells and distinct subcellular regions for precisely defined periods or using temporally patterned inputs. Nevertheless, the design of an optogenetic regulator for a new target is often a strenuous multidisciplinary task guided by combinations of crystallography, directed evolution, library screening, molecular modelling and/or mutational interweaving strategies6,7,8,9,10. It therefore remains challenging for a typical research lab to implement new optogenetic designs for new proteins and pathways.

Here we develop and evaluate a simple protocol for developing optogenetic regulators of new targets, specifically MAP kinases JNK and p38, for which the currently available tools are limited in spatiotemporal precision. We demonstrate that a LOV2-based regulator of JNK, OptoJNKi, can be applied as an optopharmacological tool without the need for gene transfer, and provide detailed protocols to successfully produce and transfer recombinant OptoJNKi into neurons. To target MAP kinases, we made use of known D-domain interactions55,56. We found AsLOV2Jα sterically constrained JNK inhibitor peptides, showing considerable dark/lit-state JNK-binding ratios for the optimal peptide (~30-fold, Fig. 1f,g). This was surprising considering reported difficulties achieving steric hindrance-based light regulation of either proteins or short peptides6,8,9. Molecular dynamic simulation suggested that phenylalanine at or near the C terminus could be ‘caged’ by a novel non-polar LOV2 domain pocket formed between residues of the Iβ-sheet and the Aβ/Bβ loop (Fig. 9). This distal pocket might cooperate with the dark-state α-helical form of the Jα sequence to constrain and thereby generate steric hindrance of fused inhibitor peptide that can be released by light, leading to LED Tube China regulation of JNK that we observed. In support of this, a point mutation in this pocket releases the JIP sequence (without disrupting protein stability as judged by soluble expression level) so that dark-state binding to JNK became indistinguishable from lit-state binding (Fig. 9d).

These structural considerations suggested a simple protocol

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  Blog créé le 16-05-2017 à 08h36 | Mis à jour le 24-06-2017 à 03h39 | Note : Pas de note