LucY: A Versatile New Fluorescent Reporter Protein.

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Fluorescent proteins are crucial tools in biological research.
• Existing fluorescent proteins have limitations in brightness and spectral properties.
• Flavin adenine dinucleotide (FAD) is a cofactor with intrinsic fluorescence, but its utility is often limited by environmental factors.

Purpose of the Study:

• To report the discovery, isolation, and characterization of a novel yellow fluorescent protein, Lucigen Yellow (LucY).
• To investigate the structural basis for LucY's enhanced fluorescence.
• To demonstrate LucY's utility as a reporter system, particularly for protein-protein interactions.

Main Methods:

• Protein purification and characterization.
• Spectroscopic analysis (excitation and emission spectra).
• High-resolution crystal structure determination.
• Bacterial expression system (E. coli) for reporter assays.
• Protein fragment complementation assays.

Main Results:

• LucY exhibits significantly enhanced fluorescence (10-fold higher than free FAD) due to FAD binding within its core.
• LucY displays excitation peaks at 276, 377, and 460 nm, and an emission peak at 530 nm, with a large Stokes shift.
• Crystal structure reveals LucY lacks a quenching aromatic residue near the FAD isoalloxazine ring, unlike related proteins.
• LucY is amenable to circular permutation and functions as a reporter for protein-protein interactions via complementation assays.

Conclusions:

• LucY is a novel, highly fluorescent protein with potential applications in various biological imaging and assay techniques.
• Its unique structural features contribute to its superior fluorescence properties.
• LucY can be engineered for protein-fragment complementation assays, enabling the study of protein-protein interactions.