Filme molecular revela como obter íons de cloreto na célula

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Bombeamento de cloreto fotoativo através da membrana celular

Bombeamento fotoativo de cloreto através da membrana celular capturado por cristalografia em série resolvida no tempo: Íons cloreto (esferas verdes) são transportados através da membrana celular pela bomba de cloreto NmHR (rosa). Crédito: Guillaume Gotthard, Sandra Mous

Pela primeira vez, um filme molecular capturou em detalhes o processo de um ânion transportado através da membrana celular por uma bomba de proteína alimentada por luz. Publicando em Ciência, os pesquisadores desvendaram o mistério de como a energia da luz inicia o processo de bombeamento – e como a natureza garantiu que não houvesse vazamento de ânions para fora.

Muitas bactérias e algas unicelulares têm bombas acionadas por luz em suas membranas celulares: proteínas que mudam de forma quando expostas a fótons, de modo que podem transportar átomos carregados para dentro ou para fora da célula. Graças a essas bombas, seus proprietários unicelulares podem se ajustar ao valor de pH ou salinidade do ambiente.

Uma dessas bactérias é Nonlabens marinus, descoberto pela primeira vez em 2012 no Oceano Pacífico. Entre outros, possui uma proteína rodopsina em sua membrana celular que transporta ânions cloreto de fora para dentro da célula. Assim como no olho humano, uma molécula da retina ligada à proteína se isomeriza quando exposta à luz. Esta isomerização inicia o processo de bombeamento. Os pesquisadores agora obtiveram informações detalhadas sobre como a bomba de cloreto Nonlabens marinus funciona.

O estudo foi liderado por Przemyslaw Nogly, que já foi pós-doutorando na PSI e agora é Ambizione Fellow e Group Leader da ETH Zürich. Com sua equipe, ele combinou experimentos em duas instalações de pesquisa em grande escala da PSI, a Swiss Light Source[{” attribute=””>SLS and the X-ray free-electron laser SwissFEL. Slower dynamics in the millisecond-range were investigated via time-resolved serial crystallography at SLS while faster, up to picosecond, events were captured at SwissFEL – then both sets of data were put together.

Mechanism of Chloride Transport Over Cell Membrane

Pink crystals reveal the mechanism of chloride transport over the cell membrane: Using time-resolved serial crystallography, the pink NmHR crystals revealed ion binding sites in the chloride transporter and pumping dynamics after photoactivation. This allowed researchers to decipher the chloride transport mechanism. Credit: Sandra Mous

“In one paper, we exploit the advantages of two state-of-the-art facilities to tell the full story of this chloride pump,” Nogly says. Jörg Standfuss, co-author of the study who built up a PSI team dedicated to creating such molecular movies, adds: “This combination enables first-class biological research as would only be possible at very few other places in the world beside PSI.”

No backflow

As the study has revealed, the chloride anion is attracted by a positively charged patch of the rhodopsin protein in Nonlabens marinus’ cell membrane. Here, the anion enters the protein and finally binds to a positive charge at the retinal molecule inside. When retinal isomerizes due to light exposure and flips over, it drags the chloride anion along and thus transports it a bit further inside the protein. “This is how light energy is directly converted into kinetic energy, triggering the very first step of the ion transport,” Sandra Mous says, a PhD student in Nogly’s group and first author of the paper.

Being on the other side of the retinal molecule now, the chloride ion has reached a point of no return. From here, it goes only further inside the cell. An amino acid helix also relaxes when chloride moves along, additionally obstructing the passage back outside. “During the transport, two molecular gates thus make sure that chloride only moves in one direction: inside,” Nogly says. One pumping process in total takes about 100 milliseconds.

Two years ago, Jörg Standfuss, Przemyslaw Nogly and their team unravelled the mechanism of another light-driven bacterial pump: the sodium pump of Krokinobacter eikastus. Researchers are eager to discover the details of light-driven pumps because these proteins are valuable optogenetic tools: genetically engineered into mammalian neurons, they make it possible to control the neurons activities by light and thus research their function.

Reference: “Dynamics and mechanism of a light-driven chloride pump” by Sandra Mous, Guillaume Gotthard, David Ehrenberg, Saumik Sen, Tobias Weinert, Philip J. M. Johnson, Daniel James, Karol Nass, Antonia Furrer, Demet Kekilli, Pikyee Ma, Steffen Brünle, Cecilia Maria Casadei, Isabelle Martiel, Florian Dworkowski, Dardan Gashi, Petr Skopintsev, Maximilian Wranik, Gregor Knopp, Ezequiel Panepucci, Valerie Panneels, Claudio Cirelli, Dmitry Ozerov, Gebhard Schertler, Meitian Wang, Chris Milne, Joerg Standfuss, Igor Schapiro, Joachim Heberle and Przemyslaw Nogly, 3 February 2022, Science.
DOI: 10.1126/science.abj6663





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