Proteins sail away with new NMR approach

Using NMR to determine protein structure has two enormous advantages over traditional crystallographic methods. The first is that it works with proteins that cannot be crystallised, but perhaps even more importantly it can reveal structural details and dynamics of proteins in their native state, whether in solution or embedded in a cell membrane. But, there is one big disadvantage. Proteins any bigger than about 25 kilodaltons have been off-limits to even the most powerful NMR spectrometer. Now, Japanese researchers have developed a technique which they claim could change all that and have demonstrated its prowess on a 41 kDa protein. Masatsune Kainosho of the Tokyo Metropolitan University and colleagues have tagged a large protein with isotopic labels with the aim of producing narrower peaks in a simplified NMR spectrum. However, rather than conventional isotopic labelling, they have developed an approach they call stereo-array isotope labelling (SAIL), which they say addresses the two-part problem that has until now meant the larger proteins were inaccessible to NMR - complex spectra with lots of overlapping signals and the "correlation-time problem" due to slow tumbling in solution and consequently broad line-widths. Stanley Opella of the University of California, San Diego, explains the SAIL system in the March 8th issue of Nature, the technique "incorporates deuterium into a protein's constituent amino acids such that each carbon or nitrogen nucleus in the final protein will have at most one 1H nucleus bonded to it, the remaining hydrogen atoms having been replaced by 2H. These amino acids are then used to make the protein in a cell-free expression system that maintains the careful positioning of the isotopes." The product is a protein with an isotopic replacement pattern that can be correlated with subsequent spectrum and so more precise information gleaned more readily. "Kainosho and colleagues' study is a significant advance in the field of protein NMR. It demonstrates that by dipping your heel in the river while sailing, the protein size problem will fade away," says Opella. Kainosho and his team have demonstrated the efficacy of this method on the 17 kDa protein calmodulin and the 41 kDa maltodextrin-binding protein. "This makes a large class of proteins newly accessible to detailed solution structure determination," Kainosho says." The team is now optimising SAIL patterns, so that they can work on even bigger proteins, perhaps up to 100 kDa. Considering that fewer than 1 in 50 of the protein NMR structures lodged with the Protein Data Bank are more than 25kDa, this approach could set protein science on a new voyage of discovery. "Source":[ http://www.spectroscopynow.com/coi/cda/detail.cda?id=12866&type=Feature&chId=5&page=1].

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