Scientific Method —

How did molecular machines evolve? Researchers resurrect lost proteins to find out

Researchers have gone back in time, calculating what the ancestral form of a …

The ring of proteins being studied, labelled C in blue, are part of a molecular machine that rotates to move protons across a membrane.

Our cells are filled with complexes that can contain dozens of proteins, all with precise interactions that ensure the complex comes together and functions in a consistent manner. These complexes, which can contain dozens of individual proteins, often have activities that mimic those of human-produced equipment, and have earned the nickname "molecular machines" accordingly.

If a molecular machine requires so many precisely positioned parts to function, how could it possibly evolve? That question has been part of a populist attack on evolution but, contrary to its proponents, scientists have a number of ideas about the evolution of this machinery. It's just that those ideas can be very hard to test, since we can't go back in time and look at the predecessors to today's machines.

Advances in DNA sequencing, however, have allowed us to calculate what the earlier proteins must have looked like. And scientists have now started to engineer DNA sequences that "resurrect" these long dead proteins, and examine how they function. In the latest work of this sort, a team has resurrected parts of an ancient molecular machine, and shown how some of its specialized protein components evolved.

The complex in question is an ATP-driven proton pump. It burns ATP, the cell's power source, and uses that energy to send protons across a membrane. In this case, the pump controls the pH of a compartment within the cell, which is necessary for survival under certain conditions. But its very similar to a far more important complex, the one that runs in reverse and harvests differences in proton concentrations to make ATP, and thus provide power to the cell.

The pump acts like a molecular machine. On one side of the membrane, it has a series of binding sites for ATP. Within the membrane, it has a collar that surrounds an inner core. The reaction that burns ATP drives the inner core to rotate a bit like an axle and, as it rotates, it carries protons from one side of the membrane to the other. Reversing the process—having protons drive the rotation in the opposite direction—leads to the production of ATP.

The researchers looked at the core of the machinery, which is made up of six proteins arranged in a circle. In fungi such as yeast, the complex is made of three types of proteins: a set of four identical ones (Anc.3), and one copy each of Anc.11 and Anc.16. All of them are essential for the protein's function. If any of the genes are knocked out, yeast will die if grown under specific conditions.

Because we have sequences for the genes that encode these proteins in a wide variety of species, it's possible to reconstruct a tree that describes their common descent, with those sequences that show the fewest differences generally being the most closely related. By doing these calculations, the authors determined that Anc.3 and Anc.11 are closely related, with Anc.11 appearing first in the ancestors of all modern fungi. This ancestral form, which the authors call Anc.3-11, had also come from a duplication of the original gene, with the other copy going on to form Anc.16.

The key thing for this work is that the analysis doesn't just tell us that Anc.3-11 once existed, it tells us what its sequence should be. And that allowed the authors to order some DNA with the right sequence, and use it to replace the genes for both Anc.3 and Anc.11. The cells didn't grow as well as the ones that had both modern versions of the gene, but the yeast continued to survive and grow.

The authors also concluded there was an advantage to having specialized versions of the gene. The evolutionary tree suggested that some changes were older than others, and the authors tried introducing a number of these to their Anc.3-11 gene. A single mutation was able to destroy the ability of Anc3.11 to function as Anc.3; that change, however, enhanced its ability to substitute for Anc.11.

The complex that currently exists forms because the three proteins mentioned here can only interact with each other in specific orientations—lose any of them, and you can't close up the complex into a neat circle that will fit at the core of the machine. The single mutation that created the specialized Anc.11 protein appears to alter a part of the protein that sits on the surface and interacts with the other parts of the ring.

This means of evolving complex molecular machines—duplication followed by specialization—had been proposed previously, but it was difficult to generate this sort of direct, biological test of the model. The ability to resurrect ancient proteins has changed that, and I expect we'll be hearing more about how evolution has assembled these machines.

Nature, 2012. DOI: 10.1038/nature10724  (About DOIs).

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