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"We studied one enzyme involved in glycosylation, the one that recognizes the protein sequence and adds the sugar chains to the protein as it is being synthesized by the cell," said William J. Lennarz of Stony Brook University, a coauthor on the paper. "The challenge is that the enzyme, known as oligosaccharide transferase (OT), is large by protein standards, has eight intricately linked components, and sits embedded in a membrane within the cell's protein-manufacturing machinery." "Membrane proteins, particularly large ones, are very difficult to study structurally," added Li. So the scientists turned to a technique called cryo-electron microscopy (cryo-EM), which shows great promise in deciphering large membrane protein structures. "We imaged the purified OT complex by cryo-EM and obtained a first snapshot of the complex by computer reconstruction of the micrographs," said Li, a cryo-EM expert. In cryo-EM, he explained, samples are frozen in vitreous ice and maintained at cryogenic temperatures (-274° Fahrenheit) using liquid nitrogen while the samples are photographed in the high vacuum of an electron microscope. The sophisticated cryo-EM machine resides in Brookhaven Lab's biology department. Li and his collaborators also measured the mass of the OT complex at Brookhaven's Scanning Transmission Electron Microscope (STEM) facility. The structure deciphered by the group helps to explain many biochemical phenomena observed about the enzyme complex over the past two decades. It also offers hints as to how the enzyme performs its various jobs, from recognizing the sugar molecules to be added to the protein, scanning the protein as it is formed to identify the sites where sugars should be attached, and transferring the sugar molecules to the protein at the right positions. "OT physically associates with the protein translocation channel which moves a protein across a membrane and the cell's protein synthesis machinery, forming an efficient three-machine assembly line for protein translation, translocation, and glycosylation," Li said. The researchers say further research is needed to illuminate the molecular mechanisms of disorders of glycosylation involving oligosaccharide transferase. For example, they would like to do structural studies of the enzyme at higher resolution in complex with substrates or in association with the cell's protein translocation and protein synthesis machinery. A new facility Brookhaven Lab hopes to begin construction on next year, known as the National Synchrotron Light Source II, would greatly increase the precision of this work.
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