Affiliation: | 1. Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung, Republic of Korea Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of Korea Contribution: Conceptualization, Methodology, Data curation, Writing - original draft, Investigation;2. Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of Korea Natural Product Research Center, Korea Institute of Science and Technology, Gangneung, Republic of Korea Contribution: Methodology, Validation;3. Department of Biochemical Engineering, Gangneung-Wonju National University, Gangneung, Republic of Korea Contribution: Writing - review & editing, Supervision;4. Department of Biological Sciences, Chungnam National University, Daejeon, Republic of Korea Contribution: Writing - review & editing, Conceptualization;5. Natural Product Research Center, Korea Institute of Science and Technology, Gangneung, Republic of Korea Department of Convergence Medicine, Wonju Severance Christian Hospital, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea Department of YM-KIST Bio-Health Convergence, Yonsei University, Wonju, Republic of Korea Contribution: Validation, Writing - review & editing, Methodology;6. Natural Product Informatics Research Center, Korea Institute of Science and Technology, Gangneung, Republic of Korea |
Abstract: | Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin-binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. Several engineering studies on CBCRs were attempted, especially for designing near-infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far-red light-emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches. |