Insight into GFPmut2 pH Dependence by Single Crystal Microspectrophotometry and X-ray Crystallography.

The fluorescence of Green Fluorescent Protein (wtGFP) and variants has been exploited in distinct applications in cellular and analytical biology. GFPs emission depends on the population of the protonated (A-state) and deprotonated (B-state) forms of the chromophore. Whereas wtGFP is pH-independent, mutants in which Ser65 is replaced by either threonine or alanine (as in GFPmut2) are pH-dependent, with a pKa around 6. Given the wtGFP pH-independence, only the structure of the protonated form was determined. The deprotonated form was deduced on the basis of the crystal structure of the Ser65Thr mutant at basic pH, assuming that it corresponds to the conformation populated in solution. Here, we present an investigation where structures of the protonated and deprotonated forms of GFPmut2 were determined from crystals grown in either MPD at pH 6 or PEG at pH 8.5, and moved to either higher or lower pH. Both crystal forms of GFPmut2 were titrated monitoring the process via polarized absorption microspectrophot...