A novel solvent-exposed analyte channel, generated by F165G substitution, on the surface of green fluorescent protein (designated His(6)GFPuv/F165G) was successfully discovered by the aid of molecular modeling software (PyMOL) in conjunction with site-directed mutagenesis. Regarding the high predictive performance of PyMOL, two pore-containing mutants namely His(6)GFPuv/H148G and His(6)GFPuv/H148G/F165G were also revealed. The pore sizes of F165G, H148G, and the double mutant H148G/F165G were in the order of 4, 4.5 and 5.5 A, respectively. These mutants were subjected to further investigation on the effect of small analytes (e.g. metal ions and hydrogen peroxide) as elucidated by fluorescence quenching experiments. Results revealed that the F165G mutant exhibited the highest metal sensitivity at physiological pH. Meanwhile, the other 2 mutants lacking histidine at position 148 had lower sensitivity against Zn(2+) and Cu(2+) than those of the template protein (His(6)GFPuv). Hence, a significant role of this histidine residue in mediating metal transfer toward the GFP chromophore was proposed and evidently demonstrated by testing in acidic condition. Results revealed that at pH 6.5 the order of metal sensitivity was found to be inverted whereby the H148G/F165G became the most sensitive mutant. The dissociation constants (K(d)) to metal ions were in the order of 4.88 x 10(-6) M, 16.67 x 10(-6) M, 25 x 10(-6) M, and 33.33 x 10(-6) M for His(6)GFPuv/F165G, His(6)GFPuv, His(6)GFPuv/H148G/F165G and His(6)GFPuv/H148G, respectively. Sensitivity against hydrogen peroxide was in the order of H148G/F165G > H148G > F165G indicating the crucial role of pore diameters. However, it should be mentioned that H148G substitution caused a markedly decrease in pH- and thermo-stability. Taken together, our findings rendered the novel pore of GFP as formed by F165G substitution to be a high impact channel without adversely affecting the intrinsic fluorescent properties. This opens up a great potential of using F165G mutant in enhancing the sensitivity of GFP in future development of biosensors.
Keywords: Green Fluorescent Protein (GFP); analyte channel; analyte sensitivity; molecular modeling; site-directed mutagenesis.