![]() ![]() Thus, for a long time, Gly67 was deemed the only residue that allows a central α-helix with a kinked conformation required for maturation of the chromophore. 11 concluded that substitution Gly67Ala impairs chromophore formation due to steric rather than conformational restrictions imposed by the side chain of Ala67 that has a significant van der Waals collision with the Thr63 carbonyl oxygen 12. A thorough direct structural study of GFP variants with non-canonical chromophore tripeptides undertaken by Barondeau et al. The attempts to replace Gly67 with the next smallest residue Ala, either destroyed the fluorescence, yielded unstable FPs 15, or prevented chromophore maturation 16. With several distinct types of chromophore chemistry and structure (e.g., avGFP, Kaede, DsRed, etc.), the third chromophore-forming residue remained unchanged. Although the second residue of the chromophore-forming triade has to be aromatic to make it fluorescent, chromophore maturation still occurs for non-fluorescent variants with Ser, Leu, and Gly in its place 4, 12– 14. Variation of the first two residues, even though the second is limited to four aromatic amino acids, resulted in a vast amount of GFP-like FPs with an emission wavelength range from 430 to 670 nm. Cyclization and dehydration take minutes to complete, whereas the rate-limiting oxidation typically takes hours 9– 10 and is, in fact, a multistep transformation, including the formation of enolate and peroxy intermediates 11– 12.įor a long time, it was believed that functional chromophore could be formed from various tripeptides, where the first amino acid residue could be any residue, the second should be an aromatic residue, and the third must be Gly 11. Chromophore maturation is a three-step process, comprising cyclization, dehydration, and oxidation 6– 8. The chromophore matures autocatalytically without the help of external cofactors or enzymes in original avGFP, it formed from the internal Ser65-Tyr66-Gly67 tripeptide. The encapsulation is doubly beneficial for the fluorescence quantum yield as it protects the chromophore from quenching by water and molecular oxygen and provides a barrier to non-radiative conformational relaxation 5. The chromophore is located in the center of the α-helix, shielded by the tightly packed strands of β-barrel. The general fold of GFP is an 11-stranded β-barrel with an internal α-helix wound around its principal axis. 3) advanced the design of fluorescent biomarkers.Įven though the GFP-like proteins are often derived from completely unrelated species and sometimes have low sequence homology, they exhibit very little variation in the tertiary structure 4. Development of new equipment and methods of imaging on the one hand and discovery of FPs with new unusual properties on the other (photoactivable FPs, photoswitchable FPs, timers, etc. To date, fluorescent biomarkers found numerous applications ranging from marking gene activity and protein labeling to tracking whole cells in tissues and GFP-based sensor applications 2– 3. Since their first biomarker application in 1994 1, many new GFP-like proteins with different fluorescence colors have been added to the arsenal of non-invasive biomarkers. Its ability to be expressed in the cells coupled with the unique autocatalytic formation of the chromophore enabled GFP use in a large number of imaging experiments in vivo in real time. The discovery of green fluorescent protein (GFP) has revolutionized the field of molecular biology and biochemistry. Both transformations are catalyzed by the same set of catalytic residues, Arg88 and Glu35-Wat-Glu211 cluster, whereas the residues in positions 62 and 102 shift the equilibrium between chromophore maturation and hydrolysis. Consideration of the protein structures revealed two alternative routes of posttranslational transformation, resulting in either chromophore maturation or hydrolysis of GYG/GYA tripeptide. Now, we have the first structure of a fluorescent protein with a successfully matured chromophore that has alanine as the third chromophore-forming residue. Until recently, it was accepted that the third chromophore-forming residue in GFP-like proteins should be glycine and efforts to replace it were in vain. Here, we report the study of four homologous lanFPs with GYG and GYA chromophores. GFP-like proteins from lancelets (lanFPs) is a new and least studied group that already generated several outstanding biomarkers (mNeonGreen is the brightest FP to date) and has some unique features. ![]()
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