Structural Characterization of Fluorescent Proteins Using Tunable Femtosecond Stimulated Raman Spectroscopy.

The versatile functions of fluorescent proteins (FPs) as fluorescence biomarkers depend on their intrinsic chromophores interacting with the protein environment. Besides X-ray crystallography, vibrational spectroscopy represents a highly valuable tool for characterizing the chromophore structure and revealing the roles of chromophore-environment interactions. In this work, we aim to benchmark the ground-state vibrational signatures of a series of FPs with emission colors spanning from green, yellow, orange, to red, as well as the solvated model chromophores for some of these FPs, using wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS) in conjunction with quantum calculations. We systematically analyzed and discussed four factors underlying the vibrational properties of FP chromophores: sidechain structure, conjugation structure, chromophore conformation, and the protein environment. A prominent bond-stretching mode characteristic of the quinoidal resonance structure is found to be conserved in most FPs and model chromophores investigated, which can be used as a vibrational marker to interpret chromophore-environment interactions and structural effects on the electronic properties of the chromophore. The fundamental insights gained for these light-sensing units (e.g., protein active sites) substantiate the unique and powerful capability of wavelength-tunable FSRS in delineating FP chromophore properties with high sensitivity and resolution in solution and protein matrices. The comprehensive characterization for various FPs across a colorful palette could also serve as a solid foundation for future spectroscopic studies and the rational engineering of FPs with diverse and improved functions.

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids.

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

Two independent routes of post-translational chemistry in fluorescent protein FusionRed.

The crystal structure of monomeric red fluorescent protein FusionRed (λex/λem 580/608 mn) has been determined at 1.09 Å resolution and revealed two alternative routes of post-translational chemistry, resulting in distinctly different products. The refinement occupancies suggest the 60:40 ratio of the mature Met63-Tyr64-Gly65 chromophore and uncyclized chromophore-forming tripeptide with the protein backbone cleaved between Met63 and the preceding Phe62 and oxidized Cα-Cß bond of Tyr64. We analyzed the structures of FusionRed and several related red fluorescent proteins, identified structural elements causing hydrolysis of the peptide bond, and verified their impact by single point mutagenesis. These findings advance the understanding of the post-translational chemistry of GFP-like fluorescent proteins beyond the canonical cyclization-dehydration-oxidation mechanism. They also show that impaired cyclization does not prevent chromophore-forming tripeptide from further transformations enabled by the same set of catalytic residues. Our mutagenesis efforts resulted in inhibition of the peptide backbone cleavage, and a FusionRed variant with ~30% improved effective brightness.