A journey back in time to a long-lost protein

How do related proteins develop different functions? This is the question investigated by a research team involving DESY and the Centre for Structural Systems Biology (CSSB).

Led by Holger Sondermann, head of the Structural Microbiology group at DESY and professor at Kiel University, the researchers reconstructed the common ancestor of two bacterial enzymes and produced the long-extinct protein in the laboratory. Their findings, now published in Science Advances, provide insights into how proteins can evolve different functions over the course of evolution.

The two enzymes studied belong to a group of proteins known as nucleases. These proteins break down certain molecules made up of the building blocks of genetic material. NrnC is an enzyme that breaks down small molecules consisting of two linked DNA or RNA building blocks. The enzyme diDNase performs a similar function, but is mainly specialised in processing molecules made up of two DNA building blocks. The researchers wanted to understand how these distinct functions evolved.

The new study builds on several years of research by the Sondermann group. The researchers compare proteins from different bacteria to determine which characteristics have remained conserved over time and which have changed. In earlier studies of a particular enzyme family, Sofia Mortensen, a scientist in the Sondermann group, identified a group of proteins that were closely related to known enzymes but processed different molecules.

The team initially tried to explain the differences between the two enzymes by introducing targeted changes into the proteins. However, the modified proteins either lost their stability or failed to perform the expected function. The researchers therefore had to take a different approach. Rather than simply comparing the proteins that exist today, they looked back at their evolutionary history.

To do this, they used a method known as ancestral sequence reconstruction. This approach uses protein sequences found in living organisms and their evolutionary relationships to predict what their common ancestors may have looked like. The team used this method to reconstruct the sequence of a protein that may have existed a very long time ago. They then produced the reconstructed protein in the laboratory and studied its properties.

X-ray crystallography can be used to determine the three-dimensional structure of a protein. For their structural studies, the researchers used beamline P11 at DESY's PETRA III X-ray source. This enabled them to investigate the structure of the reconstructed ancestral protein and examine how its structure relates to its function. The team further characterized biophysical properties of the resurrected ancestors using technologies available at the Sample Preparation and Characterisation Facility at CSSB, which is operated by the European Molecular Biology Laboratory (EMBL).

The experiments showed that the reconstructed ancestor had characteristics that lay somewhere between those of the two enzymes found today. Over the course of evolution, two distinct lineages emerged, each with a different function: one became increasingly specialised in DNA, while the other broadened its capabilities and evolved to process both DNA and RNA. The study shows that new protein functions do not necessarily arise from a single, decisive change. Instead, gradual changes to a protein over the course of evolution can play an important role.

"It is fascinating to see how proteins with different functions can evolve from a common molecular tool over such long periods of time," says Sofia Mortensen. "Our study gives us an opportunity to investigate part of this process experimentally."

Sofia Mortensen led the scientific work on the project. She characterised the two enzyme families and investigated their different structures and functions.. To reconstruct the ancestral protein sequence, the team collaborated with researchers at the National Center for Biotechnology Information (NCBI) at the US National Institutes of Health (NIH).

For the researchers, the study is primarily a starting point for further investigations. The biological role of DNA dinucleotides - small molecules consisting of two linked DNA building blocks - remains largely unknown. The group therefore plans to investigate what role these molecules play in different bacteria and how their concentration is regulated within cells. One question they are particularly interested in is whether the enzymes studied play a role in helping bacteria defend themselves against viruses.

If the mechanisms under investigation turn out to be important for key processes in bacteria, they could eventually provide new avenues for biomedical research. "But we are still a long way from that," Sondermann emphasises. "First, we need to understand what biological function these molecules actually have."

» Original publication

Source: Deutsches Elektronen-Synchrotron (DESY)