UC Irvine

Green fluorescent protein (GFP) is a widely used biomarker that demands systematical rational approaches to its structure function redesign. In this work, we mainly utilized atomistic molecular dynamics simulations to inspect and visualize internal fluctuation and coordination around chromophore inside GFP, from water to nonpolar octane solvent. We found that GFP not only maintains its β-barrel structure well into the octane, but also sustains internal residue and water coordination to position the chromophore stably while suppress dihedral fluctuations of the chromophore, so that functional robustness of GFP is achieved. Our accompanied fluorescence microscope measurements accordingly confirmed the GFP functioning into the octane. Furthermore, we identified that crucial water sites inside GFP along with permeable pores on the β-barrel of the protein are largely preserved from the water to the octane solvent, which allows sufficiently fast exchanges of internal water with the bulk or with the water layer kept on the surface of the protein. By additionally pulling GFP from bulk water to octane, we suggest that the GFP function can be well maintained into the nonpolar solvent as long as, first, the protein does not denature in the nonpolar solvent nor across the polar-nonpolar solvent interface; second, a minimal set of water molecules are in accompany with the protein; third, the nonpolar solvent molecules may need to be large enough to be nonpermeable via the water pores on the β-barrel.