Making the complex less complicated: Scientists create a simple, functional ion transporter

December 18, 2014 § Leave a comment

Researchers report the computational design, structure, and function of a transmembrane antiport protein. (Artwork conceived and produced by Nathan Joh)

Researchers report the computational design, structure, and function of a transmembrane protein. (Artwork conceived and produced by Nathan Joh)

Scientists have designed a stripped-down version of a protein that carries certain ions across a membrane. In doing so, the scientists have shown that the large, complex transporters found in all living things actually operate on simple principles. They’ve also demonstrated that we now know enough about these molecular machines to design them from scratch. The work is described in a paper just out in Science.

Natural transporters “are large and complex machines. And yet, transmembrane transport is an absolutely essential feature of cellular life so it must have evolved very early on,” says one of the scientists involved in the work, Gevorg Grigoryan at Dartmouth College. “How did such complex machines come into being?”

Grigoryan, William Degrado at the University of California, San Francisco, and others designed a bare-bones transporter, one that inserts into the lipid membrane to carry zinc or cobalt ions in one direction and protons in the opposite direction. They named their protein Rocker, after a feature engineered into the molecule that made it rock back and forth between different conformations to enable transport—similar to how natural transporters are thought to work. In Rocker’s case, it oscillated as it bound zinc ions at one end of the molecule and released them at the other

The researchers developed Rocker in two steps. First, they designed the minimalistic protein with the help of computer programs. “Because of the complicated design goals, having to balance membrane insertion, formation of the desired topology, ion binding in the membrane, and specific dynamic features of the molecule, we had to develop a novel computational design approach,” says Grigoryan, who led the computational work.

Next up was making the actual 25-amino acid protein in the lab and getting it to work as designed. The scientists showed that Rocker formed four-helix bundles in membranes, in agreement with the computational model. The amino acids within Rocker had to be precisely positioned so that the protein transported only zinc and cobalt, but not calcium, ions through it.

Every time Rocker transported a zinc or cobalt ion, it pushed three or four hydrogen ions in the opposite direction. The investigators used methods like X-ray crystallography, analytical ultracentrifugation, and nuclear magnetic resonance to study Rocker in action as it transported ions in and out of microscopic sacks made of lipids.

Although tiny in comparison to native transporters, “Rocker is essentially a reductive deconstruction of the transport process,” says Grigoryan.  By designing a minimalistic transporter protein from scratch, Grigoryan says, he and his colleagues showed that “selective transport itself does not necessarily require complex structure and demonstrated a plausible evolutionary mechanism by which transport could have originated.”

Down the road, Rocker can be used as a model system to understand the structural and thermodynamic factors for ion transport. It also can be used as a mold to design other types of transporters.

Andrei Lupas at the Max Planck Institute for Developmental Biology, who wrote the Perspective article accompanying the paper by DeGrado’s and Grigoryan’s teams, used a quote of Richard Feynman to drive home the importance of the work: “What I cannot create, I do not understand.”

Image from

Quote in the upper righthand corner as written by Richard Feynman at Caltech around 1988. Image from

Surviving in the Pacific Ocean, bacterial style

September 4, 2014 § Leave a comment

Scientists collected samples during a research cruise in October 2011 along a 2,500-mile stretch in the Pacific Ocean, from Hawaii to Samoa. The transect cut across regions with widely different concentrations of nutrients, from areas rich in iron to the north to areas near the equator that are rich in phosphorus and nitrogen but devoid of iron. [Credit: Brian Dimento, University of Connecticut]

Scientists collected samples during a research cruise in October 2011 along a 2,500-mile stretch in the Pacific Ocean, from Hawaii to Samoa.
[Credit: Brian Dimento, University of Connecticut]

Oceans cover 70 percent of the earth’s surface. Given this vast area, how do you thoroughly study how a particular organism survives in it? In a paper just out in Science, researchers analyzed how a type of cyanobacteria ekes out an existence in a 2,500-mile stretch of the Pacific Ocean. The researchers were able to measure for the first time changes in absolute protein concentrations expressed by the community of this cyanobacterium as it weathered scarcities of different nutrients. In another paper in the same issue of Science, a different group of researchers focused on a particular enzyme that helps marine cyanobacteria and other microorganisms survive under conditions of nutrient deficiency and discovered what makes the enzyme tick.

The oceans harbor “much of Earth’s biological diversity,” points out Mak Saito at the Woods Hole Oceanographic Institution who was the first author on the first Science paper. Researchers want to know how the microorganisms, which are the foundation of the marine food web and are essential to the cycling of biologically important elements, survive in oceans. The researchers want to understand how changes in carbon, phosphorus, nitrogen and other elements, caused by natural means or human activity, affect the survival of these critical microorganisms.

But Saito says experiments to analyze the effects of nutrients on marine microorganisms are difficult to do and tend to only give a glimpse of what’s going on. So Saito’s group turned to proteomic technologies because they could use them to quantitatively study the details of the biochemical changes happening in the microorganisms across the Pacific Ocean. The investigators spent a month on a ship, traveling across the Central Pacific Ocean, from Hawaii to Samoa, and collecting microbial protein samples from as deep as 1 kilometer from the ocean. The path they traveled cut through the northern regions that were rich in iron to areas near the equator that were plentiful in phosphorus and nitrogen but lacked iron. For each sample, the investigators filtered  300-800 liters of seawater over 4-6 hours through 0.2-micron filters and froze the samples.

When they got back to Woods Hole, they used two different proteomic methods to study how the protein content changed in their samples that they took from the 2,500-mile stretch of the Pacific Ocean. Saito says that previous studies identified many proteins in the oceans and their relative abundances. In contrast, the measurements he and his colleagues carried out are the first quantitative marine protein concentration measurements “in units of femtomoles of protein per liter of seawater,” he says. “By measuring the concentrations of proteins, we can map changes in the microbial biochemistry across the ocean basin.”

From their data, Saito and colleagues showed that multiple nutrient scarcities affected the cyanobacterial community they chose to track. Their conclusion refutes the notion on which previous work in the field was based, which is microbial growth and protein production was at the mercy of a single nutrient that was scarcest.

Indeed, “biogeochemists have realised that the availability of more than one inorganic nutrient may simultaneously restrict growth of microorganisms, particularly if the concentrations of the nutrients are linked by biological processes,” says Ben Berks at the University of Oxford in the U.K. who led the team in the second Science paper that identified a critical cofactor for an alkaline phosphatase found in cyanobacteria and other microorganisms. The team on the second Science paper is unaffiliated with Saito’s team on the first Science paper.

The phosphatase, PhoX, was reported to be a calcium-dependent enzyme. “However, we noticed that the purified protein had a purple color and we knew this could not arise from calcium ions,” says Berks. “This observation prompted us to investigate the nature of the PhoX cofactor.”

Although PhoX activity is critical in many microorganisms, the enzyme has not been characterized in detail.  “Possibly it reflects the fact that, although the enzyme is widespread in environmental organisms, it is not present in commonly studied model organisms,” suggests Berks.

The investigators crystallized the enzyme and then used an X-ray spectroscopic technique called micro-PIXE as well as electron paramagnetic resonance spectroscopy to identify the metals that were a part of the enzyme. They identified an iron-calcium cofactor.

Previously PhoX was thought to be a simple calcium-dependent enzyme. Calcium is abundant in seawater. If calcium was readily available, Saito says, “people wondered how microbes were maintaining the PhoA zinc alkaline phosphatase.”

Zinc is a rare commodity in marine environments. Why would a microorganism go through the trouble of relying on zinc when there was plenty of calcium to spare for enzyme activity? Now that Berks and colleagues have shown that PhoX depends on iron and calcium to function, says Saito, it now becomes clear the microorganisms are forced to make do with two different scarce elements.

The discovery of a new enzyme cofactor also means that the work of marine biochemists has a long way to go. As Berks notes, “The work demonstrates that there are still novel biological cofactors to discover within the pool of currently unstudied microbial proteins.”

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