Fluorescence Lifetime Phasor Analysis of the Decamer-Dimer Equilibrium of Human Peroxiredoxin 1

Abstract

Protein self-assembly is a common feature in biology and is often required for a myriad of fundamental processes, such as enzyme activity, signal transduction, and transport of solutes across membranes, among others. There are several techniques to find and assess homo-oligomer formation in proteins. Naturally, all these methods have their limitations, meaning that at least two or more different approaches are needed to characterize a case study. Herein, we present a new method to study protein associations using intrinsic fluorescence lifetime with phasors. In this case, the method is applied to determine the equilibrium dissociation constant (K_D) of human peroxiredoxin 1 (hPrx1), an efficient cysteine-dependent peroxidase, that has a quaternary structure comprised of five head-to-tail homodimers non-covalently arranged in a decamer. The hPrx1 oligomeric state not only affects its activity but also its association with other proteins. The excited state lifetime of hPrx1 has distinct values at high and low concentrations, suggesting the presence of two different species. Phasor analysis of hPrx1 emission lifetime allowed for the identification and quantification of hPrx1 decamers, dimers, and their mixture at diverse protein concentrations. Using phasor algebra, we calculated the fraction of hPrx1 decamers at different concentrations and obtained K_D (1.1 × 10^-24 M⁴) and C_0.5 (1.36 μM) values for the decamer-dimer equilibrium. The results were validated and compared with size exclusion chromatography. In addition, spectral phasors provided similar results despite the small differences in emission spectra as a function of hPrx1 concentration. The phasor approach was shown to be a highly sensitive and quantitative method to assess protein oligomerization and an attractive addition to the biophysicist's toolkit.

Keywords: dissociation constant; lifetime phasors; peroxiredoxin 1; protein oligomerization; spectral phasors; tryptophan fluorescence.

Figure 1

Fluorescence lifetime phasors approach and human peroxiredoxin 1 structure. (A) Phasor plot of two multiexponential decays (A,B) and a mixture of them (i). The universal circle is delimited by the red curve; τ_A and τ_B are indicated as black and blue circles, respectively; and τ_i is represented as an orange square. The phasor of τ_i is drawn from the origin, with module M and angle φ. All possible combinations of A and B fall on the segment (dashed line) between the individual components. (B) Zoom in of the A-B segment and determination of the fraction of A in i. The fading black shade indicates the amount of A along the segment. The A-i distance (Hyp) is calculated using the Pythagorean theorem as indicated by the dashed lines. The A-B distance is needed in order to calculate the fraction of components. (C) Structure of decameric hPrx1 (homology model, template PDB: 2Z9S). The homodimers forming the decamer are shown in different colors. The monomers of each homodimer are represented as different shades of the same color. (D) The dimer-dimer interface that separates upon dissociation. Tryptophan residues are represented as green sticks. The residue W86 is inside the interface, whereas W176 is outside.

Figure 2

Fluorescence properties of hPrx1 depend on its concentration. Black and red traces correspond to 20 μM and 0.5 μM hPrx1, respectively. (A) Emission spectra of hPrx1 with λ_exc 280 nm; the spectral center of mass (CM) values are indicated. (B) Phase delay (circles and diamonds) and modulation ratio (squares and triangles) plots obtained from analog frequency domain lifetime measurements at different light modulation frequencies. (C) Lorentz distributions obtained from the fit of emission lifetimes. Lifetime values obtained from the Lorentzian fit are indicated.

Figure 3

Dissociation of hPrx1 followed by fluorescence lifetime phasors. (A) Multifrequency domain (10–200 MHz) fluorescence lifetime measurements were performed at different hPrx1 concentrations (80–0.01 μM) and converted to phasor points. The colors represent different frequencies, and the multiplicity of points represent the different concentrations assayed. (B) Phasor points obtained at 103 MHz are shown. A black line connects phasor points obtained at high (80 μM) and low (0.2 μM) concentrations of hPrx1. Further dilution of hPrx1 led to signal loss and phasor displacement towards the buffer fluorescence (light blue line and point). The numbers indicate hPrx1 concentration (μM) at each point. (C) Data fitting to a decamer to dimer dissociation model. The fraction of decamer was calculated from the phasor coordinates using Equation (7), plotted against hPrx1 concentration, and fitted to the model (Equation (12)). Data are represented as the average ± standard deviation from three independent experiments.

Figure 4

Dissociation of hPrx1 followed by spectral phasors. (A) Normalized emission spectra of hPrx1 (λ_exc280 nm) at different concentrations (from red to dark violet: 0.5, 1, 2, 5, 10, and 80 µM). (B) Spectral phasor plot of the emission spectra in (A). (C) Phasor points for each emission spectrum are shown (zoom-in of (B)). The decamer–dimer transition is represented by the black line. (D) Plot of X_decamer vs. hPrx1 concentration and fitting to the dissociation model. Data are represented as the average ± standard deviation from three independent experiments.

Figure 5

Dissociation of hPrx1 studied by SEC. (A) Chromatograms obtained at different initial hPrx1 concentrations, showing the decamer at 3.9 min (green arrow) and the dimer at 5.2 min (red arrow). (B) Dissociation curve of the calculated X_decamer from the area under the curves as a function of hPrx1 concentration. Data are represented as the average ± standard deviation from two independent experiments.