Protein adaptation shows that life on early earth lived in a hot, acidic environment










"Given the ancient origin of the reconstructed thioredoxin enzymes, with some of them predating the buildup of atmospheric oxygen, we thought their catalytic chemistry would be simple, but we found that thioredoxin enzymes use a complex mixture of chemical mechanisms that increases their efficiency over the simpler compounds that were available in early geochemistry," said Julio Fernandez, a professor in the Department of Biological Sciences professor at Columbia University.

Fernandez led a team that included Columbia University postdoctoral researchers Raul Perez-Jimenez, Jorge Alegre-Cebollada and Sergi Garcia-Manyes, and graduate student Pallav Kosuri in using an assay based on single molecule force spectroscopy to measure the activity level of the thioredoxin enzymes under different pH levels.

For their experiments, the researchers used an atomic force microscope to pick up and stretch an engineered protein in a solution containing thioredoxin. They first applied a constant force to the protein, causing it to rapidly unfold and expose its disulfide bonds to the thioredoxin enzymes. The rate at which a thioredoxin enzyme snipped the disulfide bonds determined the enzyme's level of efficiency.

The study results showed that the three oldest thioredoxin enzymes -- those thought to have inhabited Earth 4.2 to 3.5 billion years ago -- were able to operate in lower pH environments than the modern thioredoxin enzymes.

"Our analysis indicates that ancient thioredoxin enzymes were well adapted to function under acidic conditions and that they maintained their high level of activity as they evolved in more alkaline environments," said Fernandez.

To measure the temperature range in which the enzymes operated, professor Jose Sanchez-Ruiz and graduate student Alvaro Ingles-Prieto from the Departamento de Quimica-Fisica at the Universidad de Granada in Spain used a technique called differential scanning calorimetry. This method measures the stability of enzymes by heating the enzymes at a constant rate and measuring the heat change associated with their unfolding.

The researchers found that the ancient proteins were stable at temperatures up to 32 degrees Celsius higher than the modern thioredoxins. The experiments showed that the enzymes exhibited higher temperature stability the older they were. The results provide evidence that ancestral thioredoxins adapted to the cooling trend of ancient oceans, as inferred from geological records.

"Our results confirm that life has the remarkable ability to adapt to a wide range of historical environmental conditions; and by extension, life will undoubtedly adapt to future environmental changes, albeit at some cost to many species," said Gaucher.

This study also showed that the experimental resurrection of ancient proteins together with the sensitivity of single-molecule techniques can be a powerful tool for understanding the origin and evolution of life on Earth.

The researchers are currently using this strategy to assess other enzymes to get a clearer picture of what life was like on Early Earth. They are also applying these tools to the field of biotechnology, where enzymes play important roles in many industrial processes. "The functions and characteristics we observed in the ancestral enzymes show that our techniques can be implemented to generate improved enzymes for a wide range of applications," added Perez-Jimenez.

### This project was supported by the National Aeronautics and Space Administration (NASA) (Award Nos. NNX08AO12G and NNA09DA78A). The content is solely the responsibility of the principal investigator and does not necessarily represent the official view of NASA.

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