State-dependent motion of a genetically encoded fluorescent biosensor.

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Imaging

Background:

• Genetically encoded biosensors offer single-cell resolution for measuring biochemical properties in vivo.
• Current biosensors function as "black boxes" with limited understanding of their structural states and dynamics.
• The structural basis for fluorescence changes in biosensors remains largely unexplored.

Purpose of the Study:

• To elucidate the structural mechanisms underlying the function of the LiLac lactate biosensor.
• To characterize the low- and high-fluorescence states of LiLac and the transitions between them.
• To provide insights for the rational design and engineering of novel fluorescent biosensors.

Main Methods:

• Utilized X-ray crystallography to determine the three-dimensional structures of LiLac in different states.
• Employed engineered high-affinity metal bridges to probe structural dynamics.
• Leveraged quantitative fluorescence-lifetime imaging to correlate structural changes with sensor output.

Main Results:

• LiLac exhibits a significant interdomain twist motion correlating with lactate binding.
• The high-lifetime state (low lactate) corresponds to a "sealed" conformation, while the low-lifetime state (high lactate) adopts a "cracked" conformation.
• Structural plasticity and interdomain dynamics are critical for LiLac's fluorescence response.

Conclusions:

• The study reveals the dynamic structural rearrangements in LiLac that enable lactate detection.
• Understanding these structure-dynamics relationships is crucial for advancing the field of genetically encoded biosensors.
• These findings provide a foundation for engineering improved biosensors with tailored properties.