Figure 1.
Structural representation of the GFP variants.
Three dimensional cartoon representation of the GFPcon and GFP14R showing the surface lysines (blue) and the surface arginines (red) as sticks which were mutated in the GFP14R. Graphical image was generated using Discovery Studio Visualizer from Accelrys Software Inc.
Table 1.
Salt-bridge and hydrogen bond interactions associated with the lysine and arginine residues in GFP.
Figure 2.
Spectral properties of the GFP variants.
A) Excitation and B) Emission spectrum of the GFPcon and GFP14R. All the amplitudes were arbitrarily normalized to a maximum value of 1.0. (au – arbitrary units).
Figure 3.
Effect of temperature on the stability of the GFP variants.
A) Protein samples were incubated at different temperatures for 30 minutes and the remaining fluorescence was measured. The fluorescence at time zero at the respective temperatures was taken into 100%. B) Protein samples were incubated at 70°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero was taken into 100%. (Error bar – Standard deviation of the three independent experiments).
Figure 4.
Effect of urea on the stability of the GFP variants.
A) Protein samples were incubated at 50°C with different concentrations of urea from 4 M to 8 M for 30 minutes and the remaining fluorescence was measured. The fluorescence at time zero at the respective urea concentrations was taken into 100%. B) Protein samples were incubated at 50°C in presence of 6 M urea for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero was taken into 100%. (Error bar – Standard deviation of the three independent experiments).
Figure 5.
Effect of pH on the stability of the GFP variants.
A) Protein samples were incubated at 60°C for 30 minutes in different pH buffer solutions and the remaining fluorescence was measured. The fluorescence at time zero at the respective buffer was taken into 100%. B) Protein samples in KCl buffer with pH 13.0 were incubated at 60°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero was taken into 100%. (Error bar – Standard deviation of the three independent experiments).
Figure 6.
Effect of ionic detergents on the stability of the GFP variants.
Protein samples were incubated with 1% SDS (A), 1% SDBS (B), 1% CTAB (C) and 1% DTAC (D) at 50°C for different time intervals and the remaining fluorescence was measured. The fluorescence at time zero at the respective detergent was taken into 100%. (Error bar – Standard deviation of the three independent experiments).
Table 2.
Half life of the GFP variants in different ionic detergents.
Figure 7.
Effect of SDS concentration on the stability of the GFP variants.
Protein samples were incubated with different concentrations of SDS at 50°C for 30 minutes and the remaining fluorescence was measured. The fluorescence at time zero at the respective SDS concentrations was taken into 100%. (Error bar – Standard deviation of the three independent experiments).
Table 3.
Comparison of salt-bridge and hydrogen bonds interactions of the GFP variants.
Figure 8.
Structural representation of the new salt-bridge interactions.
Comparison of salt-bridge interacting distances between GFPcon (left panel) and GFP14R (right panel) of (A) Lys/Arg79-Glu5, (B) Lys/Arg209-Asp216 and Lys/Arg52-Asp216, (C) Lys/Arg131-Asp103, (D) Lys/Arg3-Glu6, and (E) Lys/Arg140- Asp173. Black straight lines and green dashed lines indicate the salt-bridge and hydrogen bond interactions, respectively. Graphical image was generated using Discovery Studio Visualizer from Accelrys Software Inc.