VARPA: In Silico Additive Screening for Protein-Based Lighting Devices.

Protein optoelectronics is an emerging field facing implementation and stabilization challenges of proteins in harsh non-natural environments, such as dry polymers, inorganic materials, etc., operating at high temperatures/irradiations. In this context, additives promoting structural and functional protein stabilization are paramount to realize highly performing devices. On one hand, trial-error experimental assays based on previous knowledge of classical additives in aqueous solutions are effort/time-consuming, while their translation to water-less matrices is uncertain. On the other hand, computational simulations (molecular dynamics, electronic structure methods, etc.) are limited by the system size and time. Herein, ligand-binding affinity and atomic perturbations to create a day-fast computational method combining Vina And Rosetta for Protein Additives (VARPA) to simulate the stabilization effect of sugars for the archetypal enhanced green fluorescent protein embedded in a standard dry polymer color-converting filter for bio-hybrid light-emitting diodes is merged. The VARPA's sugar additive prediction trend for protein stabilization is nicely validated by thermal and photophysical studies as well as lighting device analysis. The device stability followed the predicted enhanced stability trend, reaching a 40-fold improvement compared to reference devices. Overall, VARPA can be adapted to a myriad of additives and proteins, driving first-step experimental efforts toward highly performing protein devices.

Supercharged Fluorescent Protein-Apoferritin Cocrystals for Lighting Applications.

The application of fluorescent proteins (FPs) in optoelectronics is hindered by the need for effective protocols to stabilize them under device preparation and operational conditions. Factors such as high temperatures, irradiation, and organic solvent exposure contribute to the denaturation of FPs, resulting in a low device performance. Herein, we focus on addressing the photoinduced heat generation associated with FP motion and rapid heat transfer. This leads to device temperatures of approximately 65 °C, causing FP-denaturation and a subsequent loss of device functionality. We present a FP stabilization strategy involving the integration of electrostatically self-assembled FP-apoferritin cocrystals within a silicone-based color down-converting filter. Three key achievements characterize this approach: (i) an engineering strategy to design positively supercharged FPs (+22) without compromising photoluminescence and thermal stability compared to their native form, (ii) a carefully developed crystallization protocol resulting in highly emissive cocrystals that retain the essential photoluminescence features of the FPs, and (iii) a strong reduction of the device's working temperature to 40 °C, leading to a 40-fold increase in Bio-HLEDs stability compared to reference devices.

Genetically Encoded Oligomerization for Protein-Based Lighting Devices.

Implementing proteins in optoelectronics represents a fresh idea toward a sustainable new class of materials with bio-functions that can replace environmentally unfriendly and/or toxic components without losing device performance. However, their native activity (fluorescence, catalysis, and so on) is easily lost under device fabrication/operation as non-native environments (organic solvents, organic/inorganic interfaces, and so on) and severe stress (temperature, irradiation, and so on) are involved. Herein, a gift bow genetically-encoded macro-oligomerization strategy is showcased to promote protein-protein solid interaction enabling i) high versatility with arbitrary proteins, ii) straightforward electrostatic driven control of the macro-oligomer size by ionic strength, and iii) stabilities over months in pure organic solvents and stress scenarios, allowing to integrate them into classical water-free polymer-based materials/components for optoelectronics. Indeed, rainbow-/white-emitting protein-based light-emitting diodes are fabricated, attesting a first-class performance compared to those with their respective native proteins: significantly enhanced device stabilities from a few minutes up to 100 h keeping device efficiency at high power driving conditions. Thus, the oligomerization concept is a solid bridge between biological systems and materials/components to meet expectations in bio-optoelectronics, in general, and lighting schemes, in particular.

Origin of acetylcholine antagonism in ELIC, a bacterial pentameric ligand-gated ion channel.

ELIC is a prokaryotic homopentameric ligand-gated ion channel that is homologous to vertebrate nicotinic acetylcholine receptors. Acetylcholine binds to ELIC but fails to activate it, despite bringing about conformational changes indicative of activation. Instead, acetylcholine competitively inhibits agonist-activated ELIC currents. What makes acetylcholine an agonist in an acetylcholine receptor context, and an antagonist in an ELIC context, is not known. Here we use available structures and statistical coupling analysis to identify residues in the ELIC agonist-binding site that contribute to agonism. Substitution of these ELIC residues for their acetylcholine receptor counterparts does not convert acetylcholine into an ELIC agonist, but in some cases reduces the sensitivity of ELIC to acetylcholine antagonism. Acetylcholine antagonism can be abolished by combining two substitutions that together appear to knock out acetylcholine binding. Thus, making the ELIC agonist-binding site more acetylcholine receptor-like, paradoxically reduces the apparent affinity for acetylcholine, demonstrating that residues important for agonist binding in one context can be deleterious in another. These findings reinforce the notion that although agonism originates from local interactions within the agonist-binding site, it is a global property with cryptic contributions from distant residues. Finally, our results highlight an underappreciated mechanism of antagonism, where agonists with appreciable affinity, but negligible efficacy, present as competitive antagonists.

Redesign of LAOBP to bind novel l-amino acid ligands.

Computational protein design is still a challenge for advancing structure-function relationships. While recent advances in this field are promising, more information for genuine predictions is needed. Here, we discuss different approaches applied to install novel glutamine (Gln) binding into the Lysine/Arginine/Ornithine binding protein (LAOBP) from Salmonella typhimurium. We studied the ligand binding behavior of two mutants: a binding pocket grafting design based on a structural superposition of LAOBP to the Gln binding protein QBP from Escherichia coli and a design based on statistical coupled positions. The latter showed the ability to bind Gln even though the protein was not very stable. Comparison of both approaches highlighted a nonconservative shared point mutation between LAOBP_graft and LAOBP_sca. This context dependent L117K mutation in LAOBP turned out to be sufficient for introducing Gln binding, as confirmed by different experimental techniques. Moreover, the crystal structure of LAOBP_L117K in complex with its ligand is reported.