CHAPTER 6: CHAPTER 6: APPLICATION OF AROMATIC TRIAZOLYL AMINO ACID SCAFFOLD AND ITS MONO- AND BIS-
1.4. Fluorescent Unnatural Amino Acids for Protein Monitoring
1.4.4. Fluorescent Unnatural Amino Acids: Microenvironment Sensitivity
Figure 1.21. Schematic presentation of solvatochroism explained by Jabolonski diagram.
Thus, solvatochromic probes have been recognised as ideal probes to study the polarity of microenvironments within a biomacromolecule and molecular gathering.
Therefore, solvatochromic fluorophores are important for investigation of protein folding and dynamics.
An α-amino acid, 6-(2-dimethylaminonaphthoyl) alanine (DANA) containing highly environment-sensitive (solvatochromic) fluorophore 6-propionyl-2- (dimethylamino)naphthalene (PRODAN, Figure 1.22) was synthesized in 2002.62 This amino acid has been utilised in peptides for monitoring peptide–protein and protein–protein interactions, where binding events often bring substrates into contact with more hydrophobic surfaces. An early study with DANA illustrated its potential by reporting the binding of a phosphoserine-containing 14-3-3-binding peptide to the target 14-3-3ζ protein.63
Figure 1.22. Some environment-sensitive fluorophores and their solvachromatic properties.
Aladan (1.142) is exceptionally sensitive to the polarity of its surroundings and can be incorporated site-selectively at buried and exposed sites, in both soluble and membrane proteins. Steady-state and time-resolved fluorescence measurements of Aladan residues at different buried and exposed sites in the B1 domain of protein G suggest that its interior is polar and heterogeneous (Figure 1.22). Cohen et al.
employed the amber suppression technique with a chemically charged tRNA bearing the DANA amino acid (“Aladan”), to probe electrostatic character of a protein at multiple sites.64 Incorporating Aladan into selected sites of Kir2.1 and Shaker potassium channels, it was concluded that Aladan was compatible with the cellular biomachinery. Moreover, substitutions in buried, aqueous, or lipid environments also allowed proper folding and functioning of the protein. As a proof of utility of Aladan to probe protein electrostatics, they incorporated an Fmoc variant of Aladan into the thermally stable IgG binding domain GB1 (∼6 kDa) by SPPS. By observing steady- state and time-resolved fluorescence data of GB1 mutants with exposed or buried Aladan, it was concluded that the interior of GB1 is polar and electrostatically heterogeneous. The ability to selectively position Aladan within specified regions of a protein makes it a valuable amino acid for gaining insight into protein electrostatics.
Latter on to improve on the concept of environment-sensitive fluorescent amino acid building blocks, fluorophores based on the dimethylaminophthalimide and dimethylaminonaphthalimide systems were developed, with the goal of increasing the fluorescence changes upon binding in target systems.65, 66 Thus, the fuluorescent amino acid, 4-(N,N-dimethylamino)-phthalimide propionic acid (4-DAPA) containing 4-(N,N-dimethylamino)-phthalimide (4-DMAP, 1.145, Figure 1.22) was synthesised by Impariali et al. and was found to have the advantage of being a closer size-mimic of tyrosine. Professor Imperiali have investigated that in comparison with DANA (aladan), 4-DAPA gave an approximately six fold enhancement in fluorescence emission upon binding with 14-3-3ζ protein when placed at the phosphoSer(−2) Tyr position in the 14-3-3-binding peptide, with a blue shift of 39 nm in the emission maximum of 570 nm.67
Peptide loading onto class II MHC proteins is regulated in antigen-presenting cells. To visualize these processes, Imperiali et al. have developed a series of novel fluorogenic probes containing the environment-sensitive amino acids 6-N,N- dimethylamino-2-3-naphthalimidoalanine (6-DMNA, 1.146, Figure 1.22) and 4-N,N- dimethylaminophthalimidoalanine (4-DAPA, Figure 1.22). Upon binding to class II MHC proteins these fluorophores show large changes in emission spectra, quantum yield and fluorescence lifetime. Peptides containing these fluorescent amino acids were found to bind specifically to class II MHC proteins on antigen-presenting cells and can be used to follow peptide binding in vivo. Using these probes they have tracked a developmentally regulated cell-surface peptide-binding activity in primary human monocyte-derived dendritic cells.68 These phthalimide- and naphthalimide- based fluorescence amino acids have also been incorporated into peptides recognizing PDZ and SH2 domains illustrating their versatility in diverse applications.69a-d
Impariali et al. have also prepared the same series of peptide with the amino acids 4-DMAP, 6-DMN, BADAN,70 dansyl and NBD (Figure 1.23). The environment- sensitive amino acid building blocks based on the dansyl fluorophore has also been exploited for probing protein interactions and protein unfolding.71 The microenvironment sensitivity of these fluorophores makes them ideal tools for studying structural dynamics in proteins. The Schultz et al. have incorporated dansylalanine into the human superoxide dismutase (hSOD) protein in yeast by using an evolved tRNA/leucyl-tRNA-synthetase pair for monitor protein unfolding phenomena in presence of guanidinium hydrochloride.72 More they have also genetically incorporated a PRODAN amino acid into the same protein to study conformational changes.73 The dansylalanine was also incorporated into the voltage sensitive domain (VSD) of the Ciona intestinalis voltage-sensitive phosphatase in HCN-A94 cells by Wang et al. They found that the dansylalanine was able to report, the conformational change of this domain in response to membrane polarization through environment-sensitive fluorescence emission.74
Figure 1.23. Solvatochromic amino acids incorporated model peptide series.
Another polarity-sensitive fluorescent unnatural amino acids L-Anap has been reported and genetically encoded by Wang et. al., to image protein modifications and activities in mammalian cells (Figure 1.24).75
Figure 1.24. Polarity-sensitive fluorescent unnatural amino acids L-Anap.
M. Sisido et al., have developed a modern technology for for screening fluorescent peptides containing multiply labeled fluorescent amino acids based on their protein binding specificity. Thus, they synthesized 8-mer peptides modified with a fluorescent several fluorescent unnatural amino acid (Figure 1.25). The peptides were mixed with an anti-FLAG antibody and an EGFR before incubation. Then the protein-binding peptides were recovered by gel filtration chromatography. Utilizing a combination of fluorescence analysis with gel filtration method they were able to quantify the binding peptides simultaneously.53
Figure 1.25. Structure of peptides modified with fluorescent amino acids.