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.