The application of nanosecond fluorometry to an allosteric protein

Abstract

The fluorescent probe 1-anilino-8-naphthalene sulfonate, when bound by the inactive form of phosphorylase b, behaves as if distributed between two different binding sites, whose bound dye emits fluorescence with decay times of 19 and 8 nanosecs. In 0.1 M glycylglycine, pH 7.0, the addition of the allosteric activator adenosine-5′-phosphate or the substrate glucose-1-phosphate results in the progressive loss of the component of longer decay time. At intermediate levels of activator the decay curves could be represented as the sum of the weighted contributions of two species, corresponding to those prevailing in the absence of activator and in excess activator. The activity of glycogen phosphorylase b, which is one of the most extensively studied allosteric enzymes, is controlled by the allosteric activator AMP (1, 2). Binding studies have shown that the binding of AMP in the absence of substrate is strongly cooperative, with a value of the Hill coefficient of 1.7 (2). Binding and kinetic studies have indicated that strong heterotropic cooperativity exists between the combination of the enzyme with AMP and either of the substrates glucose-1-phosphate and inorganic phosphate (1, 2, 3, 4). Phosphorylase b (molecular weight 185,000) consists of two equivalent subunits of molecular weight 92,500. Each subunit contains a single strong binding site for AMP(1, 2, 3). The AMP-induced activation of phosphorylase b has usually been attributed to a conformational transition, AMP being preferentially bound by the active form (1). The high degree of cooperativity of AMP binding has been attributed to the simultaneous conversion of both subunits to the active conformation upon the binding of a molecule of AMP by one subunit (1, 2). The existing kinetic studies have been interpreted as indicating that the binding of substrate likewise favors the transition to the active form, although differences of opinion exist as to the number and equivalence of the active states (1, 2, 5). However, a basic weakness of the model described above is the paucity of direct physical evidence for a conformational change. In recent years a rapid evolution of technique has made it possible to monitor the time decay of fluorescence directly (6). The technique of nanosecond fluorometry has been applied to a number of systems with the objective of analyzing complex decay curves or detecting excited state reactions (7). With the development of programs for analyzing multicomponent decay curves in terms of the lifetimes and amplitudes of the individual emitting species (8) it has become possible to apply nanosecond fluorometry to the problem of monitoring conformational transitions of proteins, and in particular, to the activator - or substrate - induced transformations of allosteric proteins. In general, when emission occurs from a collection of sources with different decay times, we have for the fluorescence intensity i (t) as a function of time, t: (1) i(t) = ∑ j α j, e, -t τ j where α j and τ j are the amplitude and decay time, respectively, corresponding to the jth component. If the number of components is small, the experimental curves of i(t) versus t may, in principle, be analyzed by the method of moments to yield the set of values of α j and τ j. In this report, we shall describe the results of the application of nanosecond fluorometry to the phosphorylase b system, using the fluorescent probe 1-anilino-8-naphthalene sulfonate (ANS). This probe, which is virtually non-fluorescent in aqueous solution, acquires an intense fluorescence when bound by a non-polar site on a protein (9, 10). In an earlier report, Seery and Anderson have found that the fluorescence intensity of ANS bound to phosphorylase b is sharply reduced in the presence of AMP or substrates. From parallel equilibrium dialysis studies, it was concluded that the greater part of the effect arose from the dissociation of ANS, although it was not possible to demonstrate a quantitative correlation (11). For this reason, it was not possible to make any statement as to the quantum yield or excited lifetime of the residual bound dye.