Cornell scientists use natural cell proteins to track molecular behavior

Cornell researchers have found a new and potentially more accurate way to see what proteins are doing inside living cells - using the cells' own components as built-in sensors.

This approach could help scientists study how molecules associate inside cells, including in viruses, and how proteins misfold in diseases like cancer and neurodegeneration.

The researchers discovered a novel way to use natural proteins produced by a cell as tiny sensors to report on their environment and interactions, without traditional invasive techniques that could interfere with a cell's normal biology and skew research results. The study was published July 1 in Nature Communications.

The method is mainly useful for understanding new biological mechanisms, such as those that could be involved in disease states like cancer or during infection. For example, one could conceivably track the assembly of a virus using this method to understand how and where its components are built within cells."

Brian Crane, the George W. and Grace L. Todd Professor in the Department of Chemistry and Chemical Biology in the College of Arts and Sciences and corresponding author on the publication

Crane, who directs the Weill Institute for Cell and Molecular Biology, and his colleagues focused on flavins, small, vitamin B2-derived molecules that can act like magnetic labels inside cells. This magnetic property makes them detectable by a technique called electron spin resonance (ESR) spectroscopy, which is like an MRI machine but measures extremely small changes and nanoscale distances. By tracking the behavior of proteins called flavoproteins, which carry flavins, researchers can detect how other molecules organize and move in living cells.

Because flavoproteins exist in many biological systems, the researchers saw a way to use them as built-in sensors. By triggering the flavin's magnetic properties with light, they could use ESR to study protein structures directly inside cells - without synthetic chemicals.

"We were studying the properties of certain flavoproteins and discovered that their magnetic spin-states were more stable than expected in cells," said Timothée Chauviré, a research associate within the Crane Lab at the Weill Institute and lead author on the study. "And from earlier work on light-sensitive proteins, we realized we could use light to trigger the signal we needed to detect these molecules using ESR."

Forcing artificial tags into cells might interfere with cellular function, but cells naturally produce flavin-containing probes, so "if you can trick the cell into making them, that is much better," Crane said.

To test their new method, the researchers studied a bacterial protein called Aer, which helps E. coli bacteria sense oxygen. Aer works with two other proteins, CheA and CheW, to transmit signals across the membrane. While these proteins have been studied before, this was the first time researchers were able to directly observe how the Aer receptor assembles inside a living cell.

"We learned that Aer forms higher-order assemblies, arrays of molecules in the membrane, that work together to amplify signals," Crane said. "These architectures are unstable and won't form outside of cells."

With ESR, the team measured the distance between the two flavins in an Aer dimer – complexes of two identical protein molecules – with angstrom-level precision, confirming not only the dimer structure but also revealing larger assemblies that form inside cells.

The researchers also developed a small engineered flavoprotein called iLOV, which can be genetically fused to other proteins to make them visible with ESR. This tool acts like a molecular tag, enabling scientists to study the structure and positioning of nearly any protein inside a living cell.

The study also demonstrated that ESR, previously mainly limited to purified proteins in test tubes, can now be used in living systems with remarkable detail.

"ESR spectroscopy is not limited to just studying purified molecules or reconstituted systems," Crane said.

The team is now adapting the method to other cell types, particularly mammalian cells, to see if they can track processes in more complex environments, he said.

Contributors to the study include Siddarth Chandrasekaran, Ph.D. '17; Robert Dunleavy, M.S. '19, Ph.D. '23; and Jack H. Freed, professor emeritus in the Department of Chemistry and Chemical Biology (A&S).

This research was supported by the National Science Foundation, the National Institutes of Health, the Weill Institute for Cell and Molecular Biology and the National Biomedical Center for Advanced ESR Technologies.