Identifing Unique Conformational Forms Of Phosphofructokinase Using Fluorescence Phasor Analysis
BIOPHYSICAL JOURNAL(2010)
Abstract
The two main parameters, phase angle and modulation, determined in frequency-domain fluorescence measurements, can be acquired with high precision. Unfortunately, when analyzing systems with complex decay mechanisms, error is often introduced by the imperfect modeling of this complexity. For questions that do not require a precise understanding of those mechanisms, the phasor approach allows a description of the system utilizing only the raw phase angle and modulation data with a corresponding improvement in reproducibility. In this investigation, we used phasor plots to describe the allosteric enzyme phosphofructokinase from E. coli (EcPFK). In our approach, we perform a direct transformation of the phase angle and modulation to the S and G function coordinates described in a Cartesian system as determined at an individual excitation modulation frequency. EcPFK contains a single tryptophan at position 311. Despite this simple composition, conventional fluorescence lifetime measurements of EcPFK exhibit complex decay behavior. The goal of this investigation is to describe the four species involved in the allosteric coupling between the substrate, fructose-6-phosphate (F6P) and the allosteric inhibitor, phosphoenolpyruvate (PEP), using the phasor approach. These four forms are: apo-EcPFK, EcPFK-F6P, PEP-EcPFK, and PEP-EcPFK-F6P). Special interest is on the ternary complex species (PEP-EcPFK-F6P) that is not considered in classic two-state models that attempt to explain the origin of allosteric behavior. The best results were obtained by exciting at 300 nm and collecting the fluorescence response at frequencies between 40 to 70 MHz. Our results show the presence of four unique conformations that correspond to the different ligated states of the enzyme. Notably, the ternary complex exhibits a unique phasor value, independent of whether it was formed by titrating the substrate followed by the inhibitor or vice versa. Supported by NIH Grant GM33216 and Welch Foundation Grant A1548.
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Key words
fluorescence,phasor,unique conformational forms
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