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Ancient Family of Retinal Proteins Brought to Light “Sight-Unseen”
摘要： Rhodopsins are an ancient class of light-sensing membrane proteins that bind retinylidene chromophores in order to form pigments that absorb visible light. Light absorption triggers isomerization of the chromophore, which is bound covalently within the transmembrane core of the protein, an event that drives protein conformational changes. Type-1 rhodopsins are also known as microbial rhodopsins, which include the well-known light-driven protein pump bacteriorhodopsin found in archeabacteria. Type-2 rhodopsins, or animal rhodopsins, include the sensory rhodopsins used as visual pigments found in most animals, including humans. Both Type-1 and -2 rhodopsins share a canonical seven-transmembrane helical polytopic structure with their amino terminal tails projecting to the topological outside of the cell membrane. Rhodopsins harness light energy to perform a vast array of functions, including pumping ions, gating channel activity, activating coupled enzyme systems, and transducing light sensation in vision. Pushkarev et al. have now reported the discovery of an entirely new group of microbial rhodopsins, opening up the potential for the elucidation of an extensive new class of rhodopsins that they term “heliorhodopsins.”
关键词： heliorhodopsins,retinylidene chromophores,light-sensing,membrane proteins,rhodopsins
更新于2025-09-23 15:21:21
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N-glycosylation state of TRPM8 protein revealed by terahertz spectroscopy and molecular modelling
摘要： TRPM8 member of the TRP superfamily of membrane proteins participates to various cellular processes ranging from Ca2+ uptake and cold sensation to cellular proliferation and migration. TRPM8 is a large tetrameric protein with more than 70% of its residues located in the cytoplasm. TRPM8 is N-glycosylated, with a single site per subunit. This work focuses on the N-glycosylation of TRPM8 channel that was previously studied by our group in relation to proliferation and migration of tumoral cells. Here, experimental data performed with deglycosylating agents assess that the sole glycosylation site contains complex glycans with a molecular weight of 2.5 kDa. The glycosylation state of TRPM8 in cells untreated and treated with a deglycosylating agent was addressed with Terahertz (THz) spectroscopy. Results show a clear difference between cells comprising glycosylated and deglycosylated TRPM8, the first presenting an increased THz absorption. Human TRPM8 was modelled using as templates the available TRPM8 and other TRPM channels structures. Glycosylations were modelled by considering two glycan structures with molecular weight close to the experiment: shorter and branched at the first sugar unit (glc1) and longer and unbranched (glc2). Simulation of THz spectra based on the molecular dynamics of unglycosylated and the two glycosylated TRPM8 models in lipid membrane and solvation box showed that glycan structure strongly influences the THz spectrum of the channel and of other components from the simulation system. Only spectra of TRPM8 with glc1 glycans were in agreement with the experiment, leading to the validation of glc1 glycan structure.
关键词： N-glycosylation,Terahertz spectroscopy,TRPM8,Molecular modelling,Membrane proteins
更新于2025-09-19 17:13:59
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Small-Angle Neutron Scattering Reveals Energy Landscape for Rhodopsin Photoactivation
摘要： Knowledge of the activation principles for G-protein-coupled receptors (GPCRs) is critical to development of new pharmaceuticals. Rhodopsin is the archetype for the largest GPCR family, yet the changes in protein dynamics that trigger signaling are not fully understood. Here we show that rhodopsin can be investigated by small-angle neutron scattering (SANS) in fully protiated detergent micelles under contrast matching to resolve light-induced changes in the protein structure. In SANS studies of membrane proteins, the zwitterionic detergent [(Cholamidopropyl)dimethylammonio]-propanesulfonate (CHAPS) is advantageous because of the low contrast difference between the hydrophobic core and hydrophilic head groups as compared to alkyl glycoside detergents. Combining SANS results with quasielastic neutron scattering (QENS) reveals how changes in volumetric protein shape are coupled (slaved) to the aqueous solvent. Upon light exposure rhodopsin is swollen by penetration of water into the protein core, allowing interactions with effector proteins in the visual signaling mechanism.
关键词： Detergent,Neutron Scattering,Hydration,GPCR,Protein Dynamics,Vision,Membrane Proteins,Energy landscape,Slaving,Rhodopsin
更新于2025-09-10 09:29:36
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Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization
摘要： The forces that stabilize membrane proteins remain elusive to precise quantification. Particularly important but poorly resolved are the forces present during a membrane protein’s initial unfolding, where the most native set of interactions are present. We developed a high-precision, atomic force microscopy assay to study the initial unfolding of bacteriorhodopsin. We discovered rapid near-equilibrium folding between the first three unfolding states that corresponded to the unfolding of 5 and 8 amino-acids respectively when using a cantilever optimized for 2-μs resolution. Interestingly, the third of these states was retinal stabilized and previously undetected despite being the most mechanically stable state in the whole unfolding pathway, supporting 150 pN for >1 min. We expect that this ability to measure the rapid and reversible dynamics in the initial unfolding of bacteriorhodopsin provides a platform for quantifying the energetics of membrane proteins under native-like conditions.
关键词： protein folding,single molecule force spectroscopy,site-specific bioconjugation,membrane proteins,atomic force microscopy
更新于2025-09-04 15:30:14
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Characterization of a DNA Aptamer for Ovarian Cancer Clinical Tissue Recognition and in Vivo Imaging
摘要： Backgrounds/Aims: Ovarian cancer is the most lethal gynaecologic malignancy and is difficult to detect early. The inefficient early diagnosis of ovarian cancer is the main contributor to its high mortality rate. Aptamers, as chemical antibodies, are single-stranded DNA or RNA oligonucleotides that target cells or molecules with high affinity. Methods: Binding ability of R13 was measured by flow cytometry analysis. Stability of R13 was tested in blood serum of an ovarian cancer patient. Internalization of R13 was verified by confocal microscope imaging. 80 cases ovarian cancer tissues, 10 cases normal ovary tissues in a microarray and 6 fallopian tube tissues were prepared for this study. R13’s target ability was further confirmed in vivo tumor models in NOD/SCID mice. Results: In this study, we found aptamer R13 bound to ovarian cancer cells with dissociation constants in the nanomolar range. Moreover, these results were further confirmed by tissue imaging. Next we demonstrated that the targets of R13 are membrane proteins and that its internalization occurs in a caveolae-mediated and clathrin-mediated manner. The target function of R13 was determined by imaging A2780 tumours in mouse models. Conclusion: These findings suggest that R13 is a promising novel tool to diagnose and deliver drugs to treat ovarian cancer.
关键词： Endocytosis,DNA aptamer,Membrane proteins,Ovarian cancer,Nude mouse,Tissue microarray
更新于2025-09-04 15:30:14