GOMEZ Gabriela Elena
congresos y reuniones científicas
Mapping accessibility in the bacterial mechanosensitive channel MscS to a small photoreactive probe
GÓMEZ G.E; WANG Y; ANISHKIN A; SUKHAREV S; DELFINO J.M
Simposio; 33rd Annual Symposium of The Protein Society; 2019
Diazirine (DZN), a water-sized photo reagent yielding highly reactive methylene carbene, probes protein regions? accessibility and packing. Here we target the homo heptameric bacterial mechanosensitive channel MscS playing a critical role in osmotic shock-driven osmolyte release. Three transmembrane helices (TM1-TM3) from all of the subunits form the barrel with a TM3-lined pore. Each subunit contributes to a large hollow cytoplasmic vestibule (cage) with seven side portals permeable to ions and small osmolytes. The available structural data raise questions about perturbations of the protein-lipid boundary and the solvent occupancy of the conductive pore forming a hydrophobic constriction (gate). MscS embedded in the inner membrane of Escherichia coli was labeled with DZN. After purification of the tagged protein and tryptic digestion, fragments were subjected to Orbitrap MS. Thus, methylation probabilities at single-residue resolution were mapped onto two crystal structures and a model of the resting state of MscS. Patterns reveal clear ?hot spots?, likely dictated by the specific chemical environment around DZN. Protein surfaces surrounded by lipids (TM1) are entirely protected from labeling, a likely consequence of the strong carbene-scavenging capacity of aliphatic chains. By contrast, interhelical crevices and many internal small voids appear labeled. Interestingly, some hydrophilic segments on the outer side of the cytoplasmic cage are reproducibly protected from labeling, suggesting their involvement in tight interactions with some cytoplasmic components. DZN labels side chains in the hydrophobic gate constriction. The rest of the pore interior is spared from labeling, supporting the notion that the pore is largely desolvated (vapor plugged).