If a pair was found that fit together, the researchers would then test the two now-combined proteins against a third protein until they found one that fit with that pair, and then the resulting three-piece structure was tested against other proteins, and so on. George Mobbs, a senior postdoctoral scholar research associate in chemistry and another co- first author of the paper, says the assembly happened in a "stepwise" fashion rather than pouring all the proteins together into a test tube at the same time, the researchers tested pairs of proteins to see which ones would fit together, like two puzzle pieces. Once that purification - which can require as much as 1,500 liters of bacterial culture to get enough material for a single experiment - was complete, the research team began to painstakingly test how the pieces of the NPC fit together. coli cells, break those cells open, and chemically purify each protein component." ![]() "We express each individual protein in E. "If you walk into the lab, you can see this giant wall of flasks in which cultures are growing," Petrovic says. Stefan Petrovic, a graduate student in biochemistry and molecular biophysics and one of the co-first authors of the papers, says the process began with Escherichia coli bacteria (a strain of bacteria commonly used in labs) that were genetically engineered to produce the proteins that make up the human NPC. To do this, they had to solve the equivalent of a very tiny 3-D jigsaw puzzle, using imaging techniques such as electron microscopy and X-ray crystallography on each puzzle piece. In their paper titled "Architecture of the cytoplasmic face of the nuclear pore," Hoelz and his research team describe how they mapped the structure of the side of the NPC that faces outward from the nucleus and into the cells' cytoplasm. Now, in a pair of papers published in the journal Science, Hoelz and his research team describe two important breakthroughs: the determination of the structure of the outer face of the NPC and the elucidation of the mechanism by which special proteins act like a molecular glue to hold the NPC together. Additionally, many viruses, including the one responsible for COVID-19, target and shutdown the NPC during the course of their lifecycles. Mutations in the NPC are responsible for some incurable cancers, for neurodegenerative and autoimmune diseases such as amyotrophic lateral sclerosis (ALS) and acute necrotizing encephalopathy, and for heart conditions including atrial fibrillation and early sudden cardiac death. ![]() Not only is the NPC central to the operations of the cell, it is also involved in many diseases. The implications of this research are potentially huge. Over the years, he has steadily chipped away at its secrets, unraveling them piece by piece by piece by piece. For more than two decades, Hoelz has been studying and deciphering the structure of the NPC in relation to its function. ![]() ![]() "It's a little like an airplane hangar where you can repair 747s, and the door opens to let the 747 come in, but there's a person standing there who can keep a single marble from getting out while the doors are open," says Caltech's André Hoelz, professor of chemistry and biochemistry and a Faculty Scholar of the Howard Hughes Medical Institute. The NPC ensures the nucleus gets the materials it needs for synthesizing RNA, while also protecting the DNA from the harsh environment outside the nucleus and enabling the RNA to leave the nucleus after it has been made. That RNA is then carried out of the nucleus so it can be used to manufacture the proteins the cell needs. Within the nucleus, DNA, the cell's permanent genetic code, is copied into RNA. The NPC's role as a gatekeeper of the nucleus means it is vital for the operations of the cell. Anything going in or out of the nucleus has to pass through the NPC on its way. The NPC, which is made of more than 1,000 individual proteins, is an incredibly discriminating gatekeeper for the cell's nucleus, the membrane-bound region inside a cell that holds that cell's genetic material. Among those pieces of machinery, and one of the most complex, is something known as the nuclear pore complex (NPC).
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