Genetically encoded fluorescent biosensors are powerful tools for live-cell imaging of metabolism and other processes. These biosensors are hybrid proteins that combine a binding component with a fluorescent protein (FP). While the separate structures of the two components are usually known, the actual structure and function of the hybrid protein is typically a black box. New work by the groups of Katherine Davis at Emory University and Gary Yellen at Harvard Medical School reveals an unexpectedly large “twist” that couples the binding of lactate with the “LiLac” biosensor to a change in fluorescence lifetime. Structures of both the bound and the apo form of the hybrid protein obtained at GM/CA@APS show an ~150° inter-domain twist between the FP and lactate-binding domains. A key β-strand “sealed” the chromophore more poorly in the low-lifetime, lactate-bound state than in the high-lifetime, apo state. The team tested this structural picture in solution by strategically substituting histidines at specific positions in the sensor. Adding Zn2+ to these mutated sensors led to the formation of stabilizing His-Zn2+-His metal bridges in either the lactate-bound or the apo state, with a corresponding change in the biosensor behavior. Understanding how the LiLac biosensor works should facilitate the engineering of other biosensors with a quantitative fluorescent readout, providing new insights into cellular metabolism. The work was funded by the National Institutes of Health, the National Science Foundation, and institutional funds from Harvard and MIT.
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Figure: A comparison of the lactate-bound (left) and lactate-free (right) LiLac biosensor; the overlay in the center illustrates the ligand-dependent twist in protein conformation. |
Citation: Rosen, PC, Horwitz, SM, Brooks, DJ, Kim, E, Ambarian, JA, Waidmann, Davis, KM, Yellen, G, "State-dependent motion of a genetically encoded fluorescent biosensor," Proc. Nat. Acad. Sci. USA 122 (10), e2426324122 (2025). DOI: 10.1073/pnas.2426324122
GM/CA @ APS
