A dynamical investigation of acrylodan-labeled mutant phosphate binding protein

The static and dynamical behavior of a fluorescently labeled mutant of the Escherichia coli periplasmic phosphate binding protein (PBP) was investigat ed through steady-state and time-resolved fluorescence spectroscopy; As a m eans of developing a biorecognition element for inorganic phosphate (P-i), alanine-197 of PBP was replaced with a cysteine, This site was then labeled with an environmentally sensitive fluorophore. The fluorescence emission o f the mutant PBP labeled with acrylodan (MPBP-AC) proved to be sensitive to micromolar concentrations of P-i, as indicated by a 50% increase in the st eady-state emission intensity. Steady-state results indicated that the labe ling protocol was specific for cys-197 only and did not label the wild-type PBP; thus, a site-selective labeling protocol was developed; Time-resolved measurements were used to determine the influence of the dynamics of MPBP- AC on the process of signal transduction, Time-resolved anisotropy measurem ents revealed that rotational dynamics were best described by a model with two independent motions: the global motion of the protein and the local mot ion of the acrylodan probe. The rates of both global and local rotational r eorientation of MPBP-AC were faster when the protein was P-i-bound rather t han P-i-free. This was a result of structural changes involving or surround ing both the P-i-binding site (global changes) and the residues in near pro ximity to the fluorescent reporter group (local changes). Recovery of the s emiangle (theta) indicated that local structural changes in MPBP-AC took pl ace when P-i was bound to the protein, Acrylodan gained mobility when MPBP- AC bound P-i, as indicated by the fact that theta increased by approximatel y 5 degrees, In addition, dynamic quenching measurements confirmed that str uctural changes occurred locally near the cys-197, Acrylodan became more ac cessible to iodide when MPBP-AC bound P-i, as demonstrated by the 35% incre ase in the value of the bimolecular quenching constant.