Bioluminescence is the natural process by which certain organisms, such as fireflies and jellyfish, produce light. This phenomenon has long intrigued scientists, who aim to harness the light-producing genes of these creatures for biomedical applications. UC Santa Cruz's Assistant Professor of Biomolecular Engineering, Andy Yeh, is at the forefront of developing artificial proteins that emit bioluminescence. These proteins could revolutionize bioimaging, diagnostics, drug discovery, and other fields. A recent paper published in the prestigious journal Chem details a new series of bioluminescent proteins designed by Yeh and his team. These proteins are small, efficient, highly stable, and capable of emitting multiple colors of light, making them ideal for real-time imaging in both cellular and animal models.

This work builds on the field of "de novo protein design"—the creation of entirely new proteins not found in nature. This approach recently earned a group of scientists, including Yeh's former post-doctoral advisor, David Baker, the 2024 Nobel Prize in Chemistry. The proteins discussed in Yeh's paper were created using deep learning-based protein design software developed by Baker’s team, along with protein structure prediction techniques from DeepMind, whose founder also shared in the Nobel Prize.

“We call this de novo protein design, because these proteins are computationally designed from scratch — it’s not found in nature and not even in the evolutionary trajectory. We demonstrated the use of the recently awarded Nobel Prize concept to build up new light-emitting enzymes, which serve as optical-based probes for biological studies,” Yeh said.

A New Tool for Imaging: Beyond Fluorescence

Fluorescence imaging is a widely used method in both research and clinical settings to study diseases, drug discovery, and other applications. However, fluorescence probes require external excitation light, which causes unwanted background light as every cell reacts to the external light. This can obscure the area of interest, making it harder to capture clear images.

In contrast, bioluminescent imaging is “excitation-free.” It relies on a chemical reaction to produce light, eliminating the background light problem and making it much more effective for imaging deep tissues, such as tumors.

This study demonstrates that the proteins developed by Yeh’s team work effectively at the molecular, cellular, and even whole animal levels, making them versatile tools for various types of research. This bioluminescence system is especially useful for non-invasive in vivo imaging, allowing researchers to study biological processes in real-time within the body without needing to extract samples.

Advancing the Ability to Visualize Biological Processes

The newly designed proteins are “orthogonal,” meaning their reaction centers are specifically tailored to interact with their light-emitting molecules, avoiding reactions with similar molecules that might be present in the system. This specificity makes these proteins compatible with other light-emitting enzymes that are already in use for biological research.

“The designed reaction is very specific, so it can be used in combination with existing light-emitting enzymes, because the enzyme recognizes a different molecule,” Yeh explained. “People already use light-emitting enzymes found in nature for a lot of biological research, and we are not reinventing the wheel. We are creating additional toolkits that work better and can be used in combination with the bioluminescence tools that the scientific community is familiar with.”

Yeh’s team has also developed a method for shifting the color of the emitted light. While most bioluminescent enzymes emit blue light, Yeh’s group has engineered a process that allows for emission of green, yellow, orange, and red light. This ability to produce a range of colors, known as “multiplexing,” is valuable for studying complex biological events, such as cancer progression, by enabling researchers to track multiple biological features simultaneously.

The Future of De Novo Protein Design

One of the key advantages of de novo proteins is their thermostability—these proteins can withstand higher temperatures without unfolding, unlike some natural bioluminescent enzymes. This stability is particularly important for practical applications like point-of-care diagnostics, where the proteins would not need to be stored at low temperatures during transport.

“We have now produced light-emitting enzymes with ideal protein folding that nature does not always need to optimize during evolution,” Yeh said. “This is the first instance in which we demonstrated that artificial light-emitting enzymes can produce enough photons in vertebrate animals for bioimaging. The computational protein design methods are becoming better and better, and so will the enzymes we design. I believe it is very true what David Baker said: this is just the beginning of de novo protein design.”

Through this pioneering work, Yeh and his team are opening new avenues for bioimaging and diagnostics, creating tools that can help researchers study biological processes with unprecedented clarity and efficiency.