Structure-function studies on signal transduction in photoactivatable cyclases

The ability to perceive and integrate environmental stimuli is essential for all living organisms. One important ambient factor is light that is sensed by a variety of photoreceptors. Many light- responsive proteins are directly connected to enzymatic functionalities and enable photo- activation or -inhibition of various cellular processes. Recently, the interest in light-triggered systems has increased significantly due to the establishment of optogenetics, which refers to the concept of genetically targeting biological systems to enable optical control of diverse processes. However, the growing demand for light controlled systems goes beyond the possibilities of naturally occurring photoreceptors. Even though substantial progress has been made in understanding the concepts of light activation in several photoreceptor families, the rational design of synthetic tools is not straight forward. Since mechanistic descriptions of signal transduction to effector domains differ even within photoreceptor families, it is obvious that a more detailed understanding of the underlying principles of sensor-effector coupling is required. To this end, I will study blue- and red-light activatable guanylate and adenylate cyclases, respectively. These represent artificial light-regulatable tools that are designed based on previous functional data of a blue-light regulatable adenylate cyclase and the evolutionary conservation of elements required for signal transduction. The identification of specific signalling elements for the closely related effector systems and the comparison of functionalities of different artificial chimeras will provide new insight into the coupling mechanism of sensor-effector modules. For a successful characterisation of these systems I will perform an interdisciplinary approach combining biochemistry with tools of structural biology. Atomic models obtained from x-ray crystallography will be functionally extended by the in-solution method of hydrogen-deuterium exchange (HDX) to obtain structural information of elements that are involved in photo-activation and signal transmission. The combined results will significantly strengthen our understanding of requirements for light- signal transduction from the point of the photoreceptor as well as the effector domain. Eventually this will enable a better understanding of the evolutionary design of light-regulatable photo- switches and support the rational design of artificial tools with application in the field of optogenetics.

The research project Structure-function studies on signal transduction in photoactivatable cyclases aimed at improving our understanding of the functional coupling between diverse sensor and effector proteins. In this context, the frequently observed modularity of different sensors coupled to a variety of directly linked effectors points towards the presence of more universal modes of cross-talk between individual modules, rather than a specific functional interaction for each combination of building blocks. Based on our efforts, the concept of sensor-effector coupling could be extended by a sensor-linker-effector mechanism, which builds upon the presence of short helical structures that connect the sensor to the effector. We showed that these elements can alter their structure in response to a signal being processed by the sensor and while the structural changes are relatively small, they can result in more than 500-fold increased activities of effectors catalyzing chemical reactions. Based on the observation of linker elements alternating between inhibiting and stimulating conformations, it is easy to appreciate how nature uses this concept to tune specific systems for the requirements of individual organisms. Without the need to modify critical amino acids involved in sensing external stimuli or residues required for the catalytic functionality of the effector domain, replacing parts of the helical linker enables a direct control over the relative stabilities of the linker structures and thereby provide an elegant mechanism to tune basal activities of the effector as well as the dynamic range of the system in response to diverse external signals, such as for example light. We then used our improved understanding of sensor-effector coupling to engineer artificial light-regulatable systems. One promising approach was the establishment of a red light-activated system that enables control over the generation of a second messenger compound involved in many cellular processes. For example, we demonstrated the direct control of locomotor activity of the model organism Caenorhabditis elegans in response to red light treatment. Similar optogenetic tools can be used to control processes in living systems with a high spatial and temporal precision in a non-invasive manner. Therefore, many interesting applications can be imagined ranging from basic cell biology to applied medicine. Negative side effects arising from the systemic administration of drugs could be prevented by a directed light activation of affected regions. In such a way, immune therapies to fight cancer, for example, could be initiated with exquisite spatial precision and could prevent toxic side-effects in the remainder of the body. While such tools are yet to be developed, our efforts to improve the understanding of how light sensing can be coupled to a variety cellular effectors has enabled scientists to approach the engineering of such systems in a more rational way.