Figure 1.
Absorbance and fluorescence spectra for Rtms5Y67F, Rtms5Y67F/H146S, Rtms5 and Rtms5H146S.
Spectra for Rtms5Y67F, (A); Rtms5Y67F/H146S, (B); Rtms5, (C) and Rtms5H146S, (D) were determined in 20 mM Tris-HCl, pH8.0 and 300 mM NaCl. Absorbance spectra are normalised at 280 nm. Absorbance (solid line), excitation (dashed line), and emission (dotted line).
Table 1.
Optical properties of Rtms5 variants and selected fluorescent proteins.
Figure 2.
Chemical structure of the chromophore in Rtms5Y67F and Rtms5Y67F/H146S.
The chemical structure of the mature chromophore is shown. Individual moieties identified in the text are labelled: (1), benzylidine; (2), methine; (3), imidazalinone; (4) glutaminyl; (5), acylimine linkage; and (6) glycyl. The location of the N- and C-termini are indicated.
Figure 3.
The effect of low pH on the absorbance spectra of Rtms5Y67F and Rtms5Y67F/H146S.
Rtms5Y67F (A) and Rtms5Y67F/H146S (B) at a protein concentration of 0.25 mg/ml in 0.1 M potassium phosphate, pH 2.3 were incubated at 21°C and the absorption spectra determined at selected time points. Rtms5 (C) and Rtms5H146S (D) at a protein concentration of 0.30 mg/ml in 0.1 M potassium phosphate, pH 2.3 were included as controls. The first absorbance scan of the incubation mixture (t0) is indicated. Relative trends (decrease or increase) in the absorbance spectra at different positions are indicated by arrows. The kinetics for changes in amount of individual absorbing species for each protein are shown (inset).
Figure 4.
The effect of GuHCl on the absorbance spectra of Rtms5Y67F and Rtms5Y67F/H146S.
Rtms5Y67F (A) and Rtms5Y67F/H146S (B) were diluted to a final protein concentration of 0.15 mg/ml in 0.1 M Tris-HCl (pH 8.0), 6 M GuHCl, and the absorption spectra determined at selected time points after incubation at 21°C. The first absorbance scan (t0) is indicated. Relative trends (decrease or increase) in absorbance are indicated by arrows. The kinetics for individual absorbing species is shown for each protein (inset).
Figure 5.
The effect of pH on the fluorescence emission and absorbance of Rtms5Y67F, Rtms5Y67F/H146S, Rtms5 and Rtms5 H146S.
Absorbance and fluorescence emission spectra were determined at different pH in buffers of constant ionic strength for (A), Rtms5Y67F; (B), Rtms5Y67F/H146S; (C), Rtms5 and (D), Rtms5 H146S. Values shown are those at the and
for each protein. Excitation was 440 nm for A and B, and 590 nm for C and D.
Table 2.
Rtms5Y67F and Rtms5Y67F/H146S data collection and refinement statistics.
Figure 6.
A schematic ribbon representation of an isolated protomer (A) of Rtms5Y67F showing the 11-stranded β-can motif typical of GFP-like proteins. The biological assembly as predicted by PISA is a 222 tetramer similar to Rtms5 (B). The chromophore is represented in stick format.
Table 3.
Rtms5Y67F chromophore contacts.
Figure 7.
The chromophore environment of Rtms5Y67F and Rtms5.
Stereoviews are shown comparing the chromophore environments and H-bonding for Rtms5Y67F (A) and Rtms5 (B). Chromophores are shown in orange (Rtms5Y67F) or blue (Rtms5). H-bonding is indicated by broken lines (corresponding distances are shown in Table 3). Waters are shown as red spheres. Two waters (W1092 and W2932) present in Rtms5Y67F but not Rtms5, that contribute to differences in H-bonding are labelled. The distance between W1092 and Cβ2 of the methine bridge of the Rtms5Y67F chromophore is 2.2 Å and highlighted by a red broken line. H-bonds between the 4-hydroxybenzylidine moiety of Rtms5 and Thr179 (water mediated) and Asn161 are not present in Rtms5Y67F.
Table 4.
Measured angles for the chromophores of Rtms5 variants and selected fluorescent proteins.
Figure 8.
Simulated annealing omit maps for the chromophores of Rtms5Y67F and Rtms5Y67F/H146S.
Alternate views are shown for the non-coplanar chromophores of Rtms5Y67F (A and B; orange) and Rtms5Y67F/H146S (C and D; green). Nearby waters (numbered red spheres) were included in the omit map calculation. The omit map calculation for the Rtms5Y67F/H146S chromophore included a nearby chloride (green sphere). The omit map indicates that the Rtms5Y67F chromophore is in the trans conformation whilst the Rtms5Y67F/H146S chromophore omit map is more ambiguous. The mesh representing the omit maps is contoured to 2.5σ. Difference maps showing the trans and cis Rtms5Y67F/H146S chromophore conformations under different occupancies are shown in Fig. S2.
Figure 9.
The chromophore model used for quantum chemical calculations.
The chromophore model is truncated at a level consistent with earlier studies of acylimine-substituted FP chromophore models. The neutral unprotonated form is shown. The protonation sites for each of the three singly protonated forms are indicated.
Table 5.
Results of quantum chemistry calculations† on neutral and singly protonated forms of the Rtms5Y67F chromophore.
Figure 10.
Chromophore contacts for Rtms5Y67F and Rtms5Y67F/H146S.
Selected contacts are shown for the chromophore of Rtms5Y67F (A) and Rtms5Y67F/H146S (B) highlighting the different positioning of the Ser69 side-chain relative to the acylimine oxygen. The charge associated with acylimine oxygen is thought to explain the existence of the 513 nm absorbing species in Rtms5Y67F.