Identifying Unique Conformations in Thermus Thermophilus Phosphofructokinase using Fluorescence Phasors

Abstract

Fluorescence frequency-domain measurements were used to study allosteric communication in phosphofructokinase from Thermus thermophilus (TtPFK). Phosph(enol)pyruvate (PEP) allosterically inhibits binding of the substrate fructose 6-phosphate (F6P). TtPFK has a much higher binding affinity for PEP and a smaller allosteric coupling between PEP and F6P compared to the other prokaryotic PFKs which makes it easier to form ternary complex in which TtPFK is bound to both F6P and PEP simultaneously. TtPFK contains no tryptophan, and the first tryptophan mutant examined was L313W. The fluorescence emission properties of L313W are responsive to the binding of ligand, with a 25% decrease in fluorescence intensity with the binding of PEP and 10% decrease with the binding of F6P. The dissociation constants for F6P and PEP and the coupling parameter are all relatively unchanged in L313W compared to wild type. The fluorescence phase and modulation obtained using the frequency-domain method of L313W TtPFK with F6P bound and PEP bound were determined. Phasor plots were constructed from the direct transformation of the phase angle and modulation to the S and G function coordinates. Our data show that the fluorescence of residue 313 responds to PEP binding much more strongly than to F6P binding. We have determined the optimum instrumental condition to be between 30 and 70MHz. If titration of a second ligand to the binary complex produces a unique phasor point, different from either binary complex and is also off the line between binary complexes, it can suggest the presence of a different conformation for the ternary complex, which is not anticipated by the traditional two-state model. Eventually tryptophans will be placed at several positions in order to map the transmission of the allosteric information. Supported by NIH grant GM033216.