(a) Direct labeling of a solvent exposed cysteine residue using a thiol-reactive agent [9, 31]. (b) Incorporation of an unnatural amino acid possessing a solvatochromic fluorophore as the side chain group via suppression of the amber stop codon. A tRNA molecule designed to recognize and read-through the amber codon is charged with the unnatural amino acid [32, 42]. (c) Expressed protein ligation involves a semi-synthetic approach typically requiring either the N- or C-terminal end of the protein to be prepared by solid phase peptide synthesis (SPPS). The chromophore can be inserted either as an amino acid building block (e.g. Fmoc-protected amino acid) during SPPS, or afterwards by labeling a side-chain residue with an appropriate electrophilic derivative of the chromophore (represented as a red circle). The peptides are then ligated to the portion of the protein construct that was expressed from a recombinant gene product [33, 95, 96].

(a) Replacement of a conserved hydrophobic/aromatic residue by a solvatochromic fluorophore. Alignment of crystal structures of the (4-DAPA)-HA (2IPK, stick representation of the peptide in light blue with 4-DAPA highlighted in orange) and HA (1JWU, stick representation in teal) peptides bound to HLA-DR1 protein (class II MHC protein, surface representation), adapted from [17]. The replacement of the conserved ligand aromatic residue that occupies the P1 pocket of the HLA-DR protein by the 4-DAPA and 6-DMNA amino acids yielded highly efficient fluorogenic probes (over 1000-fold increase in fluorescence upon binding) without significantly affecting the specificity or affinity compared to the native interaction [17]. The crystal structures illustrate the ability of the small size 4-DAPA solvatochromic amino acid to replace the tyrosine of the native ligand. The fluorogenic probes have enabled the monitoring of in vivo regulation of cell-surface peptide-binding activity of class II MHC proteins in primary dendritic cells. (b) Screening for optimal positioning of the environment-sensitive fluorophore. Top: crystal structure of the third PDZ domain of PSD-95 with a modeled bound decapeptide ligand derived from the PDZ domain-binding motif of Stargazin in stick representation (NTANRRTTPV, adapted from 1TP3). Critical residues for PDZ domain-mediated interactions (at position 0 and -2) are represented in orange with red numbering. The 4-DMAP fluorophore was inserted systematically in each non-critical position with a diaminobutyric acid linker (*: except for -3 and -1, where a diaminopropionic acid linker was used instead). The respective fluorescence increases observed upon binding to the cognate PDZ domain are presented in the bar graph. The screening approach yielded a probe with a ~90-fold fluorescence increase by insertion of the fluorophore at position -5 [84]. (c) Screening for optimal linker length between the protein backbone and the environment-sensitive fluorophore 4-DMN. Cysteine labeling agent analogues derived from 4-DMN were incorporated into monocysteine mutants of calmodulin (illustrated with S38C and E11C mutants in the calcium-bound state, 1UP5) and compared at each position for the effect of the linker length on the ability of the solvatochromic fluorophore to report changes in its local protein environment upon binding of calcium [92].

(a) Representative structures of the different families of solvatochromic fluorophores (PRODAN: 6-propionyl-2-(dimethylaminonaphthalene); NBD: 7-nitrobenz-2-oxa-1,3-diazol-4yl amine or nitrobenzoxadiazole; PyMPO: 1-(2-maleimidylethyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium methanesulfonate). The position of attachment to biomolecules, either directly or via a reactive group, are indicated by wavy lines (blue when the position is unique and green when multiple alternative anchoring positions have been developed). Points of substituent variability within the structures are indicated by EDG (electron donating group), EWG (electron withdrawing group) and X (heteroatom). (b) Summary table of the solvatochromic fluorophores properties.

(a) Folding studies. (b) Mapping of a protein local environment and/or solvatation. (c) Sensors for small molecule analytes. The sensors are generally designed by exploiting protein domains that intrinsically bind a select analyte resulting in fluorescence change due to a conformational change in the protein or the displacement of the fluorophore (illustrated here with the d-glucose/d-galactose-binding protein from E. Coli, 2FW0 for the apo state and 2FVY for the glucose-bound state). (d) Reporting protein structural changes. In response to a signaling event, the protein of interest will undergo a conformational change that corresponds to a different functional state, which may be monitored by an appropriately positioned solvatochromic fluorophore (illustrated with calmodulin, 1DMO apo state and 1UP5 calcium-bound state). (e) Fragment- or peptide-based probes for the monitoring of protein interactions. A minimal fragment of one of the binding partners can be labeled with a solvatochromic fluorophore to report interactions. In the case of transient interactions, the signaling event that will induce the interaction can affect either one of the partners thus providing information on function or activity. This approach is illustrated here with the Crk SH2 domain (1JU5) that binds to phosphopeptides sequences and that can be used to report either the activity of a kinase or the phosphorylated state of a substrate. (f) Reporting protein-protein interactions. A similar approach to panel (e) can be applied to full-length proteins instead of fragments. In this case advantage can be taken of the larger interaction interface between two proteins compared to peptide-based probes that do not adopt secondary and tertiary structures.

The origin of solvatochromic effects on fluorescence.