Rational design of a circularly permuted flavin-based fluorescent protein

In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering.PMID:38356332 | DOI:10.1002/cbic.202300814

Source: Chembiochem - February 15, 2024 Category: Biochemistry Authors: Nolan T Anderson Jason Xie Asish N Chacko Vannie Liu Kang-Ching Fan Arnab Mukherjee Source Type: research