Flexibility of the molecular forms of acetylcholinesterase measured with steady-state and time-correlated fluorescence polarization spectroscopy

Abstract

Steady-state and time-correlated fluorescence polarizations have been examined for selected complexes and covalent conjugates of the 11S and (17 + 13)S forms of Torpedo acetylcholinesterase. The 11S form exists as a tetramer of apparently identical subunits, whereas the (17 + 13)S forms contain two or three sets of tetramers, disulfide-linked to an elongated collagen-like tail unit. Pyrenebutyl methylphosphonofluoridate and (dansylsulfonamido)pentyl methylphosphonofluoridate were conjugated at the active center serine whereas propidium was employed as a fluorescent ligand for the spatially removed peripheral anionic site. Steady-state polarization of the pyrenebutyl conjugates indicates rotational correlation times of approximately 400 ns for the 11S species and greater than 1100 ns for the (17 + 13)S species. Hence, the tail unit severely restricts rotational motion of the catalytic subunits. Time-correlated fluorescence polarization analysis of the 11S species indicates multiple rotational correlation times. Anisotropy decay of the propidium complex (.tau. = 6 ns) occurs in exponential manner with a rotational correlation time of .apprx. 150 ns, while covalent adducts at the active center exhibit rotational correlation times .gtoreq. 300 ns. Anisotropy decay of the (dansylsulfonamido)pentyl conjugate (.tau. = 16 ns) appears exponential with a correlation time of approximately 320 ns, whereas decay of the pyrenebutyl conjugate (.tau. = 100 ns) is described by two correlation times, .vphi.S = 18 ns and .vphi.L = 320 ns, of small (15%) and large (85%) amplitudes, respectively. Two limiting models have been considered to explain the results. The first model considers the 11S form to behave as a rigid ellipsoid of revolution, whereas the second model incorporates the capacity for segmental motion. The biphasic decay seen with the longer lived probe and the differences in anisotropy decay seen for propidium and the active site ligands support the second model which incorporates protein flexibility.