A Chemical Strategy for Amphiphile Replacement in Membrane Protein Research.

Membrane mimics are indispensable tools in the structural and functional understanding of membrane proteins (MPs). Given stringent requirements of integral MP manipulations, amphiphile replacement is often required in sample preparation for various biophysical purposes. Current protocols generally rely on physical methodologies and rarely reach complete replacement. In comparison, we report herein a chemical alternative that facilitates the exhaustive exchange of membrane-mimicking systems for MP reconstitution. This method, named sacrifice-replacement strategy, was enabled by a class of chemically cleavable detergents (CCDs), derived from the disulfide incorporation in the traditional detergent n-dodecyl-ß-d-maltopyranoside. The representative CCD behaved well in both solubilizing the diverse α-helical human G protein-coupled receptors and refolding of the ß-barrel bacterial outer membrane protein X, and more importantly, it could also be readily degraded under mild conditions. By this means, the A2A adenosine receptor was successfully reconstituted into a series of commercial detergents for stabilization screening and nanodiscs for electron microscopy analysis. Featured by the simplicity and compatibility, this CCD-mediated strategy would later find more applications when being integrated in other biophysics studies.

The structure-based traceless specific fluorescence labeling of the smoothened receptor.

The smoothened receptor (SMO) mediates the hedgehog (Hh) signaling pathway and plays a vital role in embryonic development and tumorigenesis. The visualization of SMO has the potential to provide new insights into its enigmatic mechanisms and associated disease pathogenesis. Based on recent progress in structural studies of SMO, we have designed and characterized a group of affinity probes to facilitate the turn-on fluorescence labeling of SMO at the ε-amine of K395. These chemical probes were derived from a potent SMO antagonist skeleton by the conjugation of a small non-fluorescent unit, O-nitrobenzoxadiazole (O-NBD). In this context, optimal probes were developed to be capable of efficiently and selectively lighting up SMO regardless of whether it is in micelles or in native membranes. More importantly, the resulting labeled SMO only bears a very small fluorophore and allows for the recovery of the unoccupied pocket by dissociation of the residual ligand module. These advantages should allow the probe to serve as a potential tool for monitoring SMO trafficking, understanding Hh activation mechanisms, and even the diagnosis of tumorigenesis in the future.

Multi-channel feature fusion attention Dehazing network.

Haze is a typical weather phenomena that has a significant negative impact on transportation safety, particularly in the port, highways, and airport runway areas. A multi-scale U-shaped dehazing network is proposed in this research, which is based on our multi-channel feature fusion attention structure. With the help of the feature fusion attention techniques, the model can focus on the intriguing locations with higher haze concentration area. In conjunction with UNet, it can achieve multi-scale feature reuse and residual learning, allowing it to fully utilize the feature information of each layer for image restoration. Experimental resulsts show that our technique performs well on a variety of test datasets. On highway data sets, the PSNR / SSIM / L∞ error performance over the novel technique is increased by 0.52% / 0.5% / 30.84%, 4.68% / 0.78% / 26.19% and 13.84% / 9.05% / 55.57% respectively, when compared to DehazeFormer, MIRNetv2, and FSDGN methods. The findings suggest that our proposed method performs better on image dehazing, especially in terms of L∞ error performance.

A structurally guided dissection-then-evolution strategy for ligand optimization of smoothened receptor.

We present herein a novel dissection-then-evolution strategy for ligand optimization. Using the co-crystal structure of the smoothened receptor (SMO) as a guide, we studied the modular contribution of LY2940680 by systematically "silencing" the specific interaction between the individual residue(s) and the fragment in the ligand. Following evolution by focusing on the benzoyl part finally yielded an improved ligand 21.

Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand.

The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs.