A milestone in the future development of novel red-light regulated optogenetic tools for targeted cell stimulation has been achieved by a team of scientists from Graz University of Technology.
For the first time ever, they were able to functionally characterize the three-dimensional interaction between red-light receptors and enzymatic effectors on a molecular level.
The aim of optogenetics is to control genetically modified cells using light. The architecture and composition of the linker element connecting the sensor and effector is very important in light regulation.
Helical Light Switch
To survive, cells and organisms have to adapt to new environmental conditions. This is the job of protein “building blocks” that interact with each other in different ways, thus creating cellular networks allowing adaptations to be made to changed environments.
The sensors or “receptors” of external stimulation, such as light, are at least in part coupled to specific effectors in order to specifically activate or inhibit cellular signal molecules depending on need.
“By using a combination of X-ray structural analysis and hydrogen-deuterium exchange by which the structural dynamics and conformational changes can be analysed, we managed to better understand the functional characteristics of this helical coupling element. We were able to show that illuminating the sensor with red light resulted in a rotation-like change in the coiled coil linker region, which in turn effects the enzymatic activity of the neighbouring effector.”
The Graz researchers were thus able to determine structural details of a red-light regulated full-length system and describe molecular mechanisms of signal transduction.
Rational Protein Design
The research contributes to better understanding the modularity of naturally occurring protein domains and being able to develop new optogenetic tools. Diverse combinations of different sensor modules are found in nature, such as red-light sensors, blue-light sensors and pH sensors—sometimes with identical and sometimes different effectors.
From this, the researchers conclude that there are molecular similarities in signal transduction and therefore that rational and completely arbitrary combinations of sensors and effectors which do not occur in nature are conceivable.
Andreas Winkler says,
“We are currently limited to naturally occurring systems to a great extent in the use of directly regulated enzymatic functionalities. The long-term aim is to generate new light-regulated systems which can overcome the limitations of nature and which would be of great interest for different applications in optogenetics.”
“Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light–sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate–producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light–regulated optogenetic tools.”