Bioluminescence is the natural chemical process of creating light in some living creatures that makes fireflies sparkle and some jellyfish glow. Scientists have long wanted to borrow the secrets of these animals’ light-producing genes to create similar effects in vertebrates, for various biomedical applications.

Andy Yeh, assistant professor of biomolecular engineering at UC Santa Cruz, is designing fully artificial proteins that produce bioluminescence to serve as a noninvasive method for bioimaging, diagnostics, drug discovery, and more. A new paper published in the flagship journal Chemistry reports a new series of bioluminescent proteins designed by Yeh and his group, which are small, efficient, very stable, and can produce multiple colors of light for real-time imaging in cellular and animal models.

This area of ​​designing proteins not found in nature, called “new Protein Design”, recently won a group of scientists the 2024 Nobel Prize in Chemistry, including David Baker, Yeh’s postdoctoral advisor. The proteins described in this article were created using learning protein design software deep developed by Baker’s group, as well as protein structure prediction methods produced by DeepMind, whose founder also shared the Nobel Prize.

“We call this new protein design, because these proteins are computer designed from scratch – they are not found in nature or even in the evolutionary trajectory. We demonstrated the use of the recently Nobel Prize-winning concept to create novel light-emitting enzymes, which serve as optical probes for biological studies,” Yeh said.

Better imaging probes beyond fluorescence

Many researchers and clinicians use fluorescence imaging techniques to understand diseases, contribute to drug discovery, and more. Fluorescent probes require external excitation light to glow. When external light shines on a tissue, each cell responds, creating a lot of background light that makes it more difficult to distinguish what the researcher or clinician is looking for.

Bioluminescent imaging is different in that it is “excitation-free”: the entire process of emitting light occurs at the level of a chemical reaction. Bioluminescence creates no response to background light, making it much more effective for imaging features that may be deep within a tissue, such as a tumor.

This paper shows that the researchers’ light-emitting proteins work at the level of molecules, cells and within a whole animal, making them more generalizable to different types of scientific research. It is particularly suitable for non-invasive in vivo imaging because it can reveal real-time information about deep biological processes within a tissue without having to take a sample from the body.

View multiple biological events

These specially designed proteins are “orthogonal,” with their reaction center highly matched to the shape of the designed light-emitting molecule. This means that the synthetic enzyme does not react with other similar molecules that a researcher or clinician might be using at the same time.

“The designed reaction is very specific, so it can be used in combination with existing light-emitting enzymes, because the designer enzyme recognizes a different molecule,” Yeh said. “People already use naturally occurring light-emitting enzymes for a lot of biological research, and we’re 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 and his team also developed a method to change the color of light emitted by proteins. Typically, these enzymes emit blue light, but through an efficient energy transfer process, researchers have made it possible to emit green, yellow, orange and red light. This would allow a researcher or clinician to monitor a variety of different biological features, called “multiplexing,” which are important for studying complex processes such as cancer development.

The new age of new protein design

New The proteins are highly thermostable, meaning they will not expand at high temperatures, unlike other natural bioluminescent enzymes. A highly stable protein would be much easier to use for point-of-care diagnostics because it would reduce the need for specialized low-temperature 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 time we have demonstrated that artificial light-emitting enzymes can produce enough photons in vertebrate animals for bioimaging. Computational methods for designing proteins are getting better and better, and so are the enzymes we design. I think what David Baker said is very true: this is just the beginning of a new protein design.