Study illuminates new design strategies for optogenetic tools

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Optogenetics, a technology that combines genetics and optics, uses light for the precise control of biological processes that can be as complex as the behaviour of animals.

The ability to control intrinsically light-insensitive biological processes by light has sparked excitement among researchers by providing a never-before-seen level of insight into the working mechanism of biological systems.

The design of optogenetic tools has developed rapidly over recent years. Specialized, naturally occurring, light-sensing proteins, called photoreceptors, which are used by organisms to respond to sunlight, provide the foundation for the design of these tools.

Photoreceptors consist of different building blocks (called domains) that are each equipped with a specialized function. Light absorption by the light-sensing part causes rearrangements within the photoreceptor that are transmitted to a part of the protein that can bind to DNA or perform another type of biological activity.

To create synthetic optogenetic tools with new functionalities, the light-sensing part of a photoreceptor needs to be coupled with the domain of another protein that performs the desired biological function, allowing regulation of this process by light. Rational design of such tools is difficult, however, since the different parts need to be connected in a way that allows the light signal to be transmitted efficiently to the part that performs the biological function.

Now, a new study led by the Max Planck Institute for Medical Research in Heidelberg, Germany, provides important information for the design of new robust and tightly controllable molecular tools for use in optogenetics. The study is published in the journal eLife.

“The rational design of novel light-regulated systems for optogenetic use requires understanding of the working mechanism of natural photoreceptors,” says lead author Udo Heintz.

“By studying these photoreceptors, we can identify specific principles used to sense and translate the information transported by light into biological activities. Based on this knowledge, new synthetic light-controllable proteins can be engineered to allow precise control of specific processes in living cells and freely moving animals.”

For their study, Heintz and co-author Ilme Schlichting looked into the structural changes that blue light causes in the Aureochrome 1a photoreceptor found in the alga Phaeodactylum tricornutum (PtAu1a) and how they affect the protein’s DNA-binding.

For light sensing, PtAu1a employs a module called light-oxygen-voltage (LOV) domain. Blue light activates this domain and causes structural changes that, in the case of PtAu1a, are transmitted to a part of the protein that can bind to DNA.

Heintz and Schlichting determined the three-dimensional structures of the LOV domain of PtAu1a in its inactive and light-activated state. They combined their structural studies with biophysical methods, providing detailed information on how Aureochrome 1a works.

“The limited number of protein structures that show photoreceptors in their light-activated state hampers our understanding of their working mechanism. Trapping photoreceptors in this state is a major obstacle for structural studies, as most of them revert rapidly to their inactive conformation,” Heintz explains.

The researchers found that in the inactive state (without illumination), the LOV light-sensing domain interacts directly with the DNA-binding part, hindering its binding function. When exposed to light, the light-sensing LOV domain is activated, releases the DNA-binding part and binds to the LOV domain of a second molecule. This ultimately increases PtAu1a’s ability to bind DNA at a specific target sequence.

These studies provide new structural and functional insights into the light-dependent DNA-binding of Aureochromes, contributing to the fundamental understanding of light-signaling in LOV photoreceptors. They also offer new design strategies for future Aureo-LOV-based photosensors for use in optogenetics.

“Moreover, the structural findings explain the underlying working mechanism of recently engineered, light-activatable receptor tyrosine kinases, which have potential applications in drug development,” Heintz adds.