Improving the Second-Order Nonlinear Optical Response of Fluorescent Proteins: The Symmetry Argument

We have successfully designed and expressed a new fluorescent protein with improved second-order nonlinear optical properties. It is the first time that a fluorescent protein has been rationally altered for this particular characteristic. On the basis of the specific noncentrosymmetry requirements for second-order nonlinear optical effects, we had hypothesized that the surprisingly low first hyperpolarizability (β) of the enhanced yellow fluorescent protein (eYFP) could be explained by centrosymmetric stacking of the chromophoric Tyr66 and the neighboring Tyr203 residue. The inversion center was removed by mutating Tyr203 into Phe203, with minor changes in the linear optical properties and even an improved fluorescence quantum yield. Structure determination by X-ray crystallography as well as linear optical characterization corroborate a correct folding and maturation. Measurement of β by means of hyper-Rayleigh scattering (HRS) as well as their analysis using quantum chemistry calculations validate our hypothesis. This observation can eventually lead to improved red fluorescent proteins for even better performance. On the basis of the specific function (second-harmonic generation), the color of its emission, and in analogy with the “fruit” names, we propose SHardonnay as the name for this Tyr203Phe mutant of eYFP