Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein

The fluorescence emission of yellow fluorescent proteins (YFPs) has been sh own to respond rapidly and reversibly to changes in the concentration of so me small anions such as halides; this allows for the use of YFPs as genetic ally encodable Cl- sensors that may be targeted to specific organelles in l iving cells. Fluorescence is suppressed due to protonation of the chromopho re upon anion binding, with a stronger level of interaction at low pH value s. At pH 6.0, the apparent dissociation constant (K-app) for Cl- is 32 mM f or YFP and 22 mM for YFP-H148Q, whereas at pH 7.5, K-app is 777 mM and 154 mM, respectively. In the cytosol, YFP-H148Q appears most promising as a hal ide sensor due to its high degree of sensitivity towards I- (K-app = 23 mM at pH 7.5). To aid in the design of variants with improved levels of specif icity and affinity for Cl-, we solved apo and I--bound crystal structures o f YFP-H148Q to 2.1 Angstrom resolution. The halide-binding site is found ne ar van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stab ilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispen sible for tight binding. A series of conformational changes occurs in the a mphiphilic site upon anion binding, which appear to be propagated to the be ta-bulge region around residue 148 on the protein surface. Anion binding ra ises the chromophore pK(a) values, since delocalization of the phenolate ne gative charge over the chromophore skeleton is suppressed. Extraction of mi croscopic binding constants for the Linked equilibrium between anion and pr oton binding indicates that anion selectivity by YFP is related to hydratio n forces. Specific suggestions to improve Cl- binding to YFP-H148Q based on size and hydration energy are proposed. (C) 2000 Academic Press.