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New Method of Protein Structure Elucidation with Mutant Genes

“Early indications suggest the technique can solve structures with only a fraction of all possible mutants. The approach could even work using measures of stability for proteins that lack known binding partners.”
New Method of Protein Structure Elucidation with Mutant Genes

Image Courtesy: Science magazine. Image for representational use only.

Deciphering protein structure in its 3D form is of immense importance for understanding the function the protein performs inside a cell. A change in its structure can lead to an alteration in its function. That’s how the important stream of structural biology emerged and developed. But the conventional methods applied in this stream of research require expensive facilities with super cooled, powerful magnets or stadium size synchrotrons.

Now, two teams working independently have reported to come out with a new technique of protein structure elucidation. This new technique is a combination of genetic and biochemical tools that can reveal protein structure in their natural condition, while they do their work inside cell. The previous methods visualise proteins in crystals or solutions; there had been no method of visualising protein in real time.

The common approach that structural biologists of today adopt in determining a protein’s structure requires making a crystal of the protein, and then blasting it with X ray and tracking the position and identity of each atom. Apart from it, other techniques like NMR spectroscopy, cryoelectron microscopy that are in regular use, need huge amount of proteins and also could take months for completion. Till now, researchers have determined the structures of about 1, 50,000 proteins out of millions thought to exist and this process has already taken decades of efforts.

In an effort to speed up the process, some researchers have tried to predict the most likely structure of a protein simply from the sequence of amino acids and the probable interactions among the atoms. Notably, this is a computational approach and the accuracy of such computations more often lags behind experimental methods. In one recent effort, comparison of the same protein in multiple species was done. This was to find pairs of amino acids that have evolved together even though they are far apart on protein’s linear sequence.

The New Technique

Two separate teams, led by Debora Marks, Harvard Medical School, Boston and Ben Lehner, Barcelona Institute of Science and Technology, Spain, have reported separately in Nature Genetics to have developed the new technique of protein structure determination. The idea that they banked upon is to find out interacting amino acids within a protein by systematically mutating each amino acids and tracking how the changes alter the function of the protein, for example binding to another molecule.

Both the groups started from what Ren Sun of University of California, Los Angeles did in 2014. Sun’s team reported that they have created more than half a million copies of the bacterial gene GB1. Each of the protein copies had mutant amino acids. In the single mutants the researchers changed each amino acids for one of the 19 other options. In the double mutants they changed pair of amino acids in nearly all possible combinations.

Marks’s and Lehner’s groups tried to combine the binding data of the single and double mutants to determine which amino acids amino acids interact most strongly and are therefore likely sit next to each other in the protein’s 3D structure. After tracking the occurrence of many such mutants, they fed it to a structure prediction programme. The team could compute the shape of the main backbone of GB1. Moreover, they could manage to have their computation of the structure to a resolution similar to already known X ray structure. They have reported their success in other small proteins and RNA as well.

“Early indications suggest the technique can solve structures with only a fraction of all possible mutants. The approach could even work using measures of stability for proteins that lack known binding partners”—the teams expressed their optimism.

“It’s fantastic, the new approach doesn’t offer the full atomic map that standard approaches do, but the general shape it provides for a protein nonetheless offers “extremely valuable” clues to its function. It could have a really big impact on efforts to determine structures of proteins that are membrane-bound or part of large complexes, both of which are difficult to study with standard methods,” says Douglas Fowler, a genome scientist at the University of Washington in Seattle.

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