Long- and Short-Range Electrostatic Fields in GFP Mutants: Implications for Spectral Tuning.

The majority of protein functions are governed by their internal local electrostatics. Quantitative information about these interactions can shed light on how proteins work and allow for improving/altering their performance. Green fluorescent protein (GFP) and its mutation variants provide unique optical windows for interrogation of internal electric fields, thanks to the intrinsic fluorophore group formed inside them. Here we use an all-optical method, based on the independent measurements of transition frequency and one- and two-photon absorption cross sections in a number of GFP mutants to evaluate these internal electric fields. Two physical models based on the quadratic Stark effect, either with or without taking into account structural (bond-length) changes of the chromophore in varying field, allow us to separately evaluate the long-range and the total effective (short- and long-range) fields. Both types of the field quantitatively agree with the results of independent molecular dynamic simulations, justifying our method of measurement.