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.
Stable/efficient low‐energy emitters for photon down‐conversion in bio‐hybrid light‐emitting diodes (Bio‐HLEDs) are still challenging, as the archetypal fluorescent protein (FP) mCherry has led to the best deep‐red Bio‐HLEDs with poor stabilities: 3 h (on‐chip)/160 h (remote). Capitalizing on the excellent refolding under temperature/pH/chemical stress, high brightness, and high compatibility with polysaccharides of phycobiliproteins (smURFP), first‐class low‐energy emitting Bio‐HLEDs are achieved. They outperform those with mCherry regardless of using reference polyethylene oxide (on‐chip: 24 h vs. 3 h) and new biopolymer hydroxypropyl cellulose (HPC; on‐chip: 44 h vs. 3 h) coatings. Fine optimization of smURFP‐HPC‐coatings leads to stable record devices (on‐chip: 2600 h/108 days) compared to champion devices with perylene diimides (on‐chip: <700 h) and artificial FPs (on‐chip: 35 h). Finally, spectroscopy/computational/thermal assays confirm that device degradation is related to the photo‐induced reduction of biliverdin to bilirubin. Overall, this study pinpoints a new family of biogenic emitters toward superior protein‐based lighting.
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.
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