Linear Combination Properties of Spectral Phasors Allow Thermodynamic Analysis of Reversible Denaturation of Multidomain and Membrane Proteins
4 affiliations
- 1Laboratorio de Biofísica Molecular, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires—CONICET, Buenos Aires 1113, Argentina
- 2Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Buenos Aires 1113, Argentina
- 3Unidad de Bioimagenología Avanzada, Institut Pasteur de Montevideo and Universidad de la República, Montevideo 11400, Uruguay
- 4Unidad Académica de Fisiopatología, Hospital de Clínicas, Facultad de Medicina, Universidad de la República, Montevideo 11600, Uruguay
Abstract
Denaturation of small globular proteins is often described as a reversible two-state transition between native and denatured conformations. Conversely, multidomain and membrane proteins frequently populate several partially folded intermediate states during denaturation. Traditional thermodynamic analysis of fluorescence intensity often fails to reliably identify these intermediate states. Alternatively, spectral phasors provide an effective model-free way to monitor these complex conformational changes by resolving overlapping fluorescence signals with small differences in the position or shape of the spectra. Nevertheless, converting phasor trajectories into quantitative thermodynamic information remains challenging when quantum yields do not remain constant during a given process, thus limiting their application to protein stability studies. In this work, we introduce a thermodynamic framework for quantifying protein stability from phasor trajectories. We validate this formalism using simulated spectral data generated from two- and three-state transitions, including changes in fluorescence quantum yields, successfully recovering the thermodynamic parameters used to generate the simulations. Additionally, we applied this approach to analyze published data on urea-induced unfolding of E. coli Outer Membrane Protein A. Phasor analysis identified a two-state transition, and the thermodynamic parameters agreed well with those obtained from total fluorescence intensity, supporting the validity of the proposed approach. Finally, applying this framework to the SDS-induced denaturation of Archaeoglobus fulgidus Cu(I)-transport ATPase enabled a complete thermodynamic characterization of the three transitions identified by phasor analysis of intrinsic fluorescence, which could not be resolved using fluorescence intensity alone. This formalism establishes fluorescence spectral phasors as a powerful quantitative approach for protein stability analysis.
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BibTeX
@article{angelini2026linear,
title = {{Linear Combination Properties of Spectral Phasors Allow Thermodynamic Analysis of Reversible Denaturation of Multidomain and Membrane Proteins}},
author = {Alvaro A. Recoulat Angelini and Leonel Malacrida and F. Luis González Flecha},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27209233},
url = {https://www.mdpi.com/1422-0067/27/20/9233},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Linear Combination Properties of Spectral Phasors Allow Thermodynamic Analysis of Reversible Denaturation of Multidomain and Membrane Proteins
AU - Alvaro A. Recoulat Angelini
AU - Leonel Malacrida
AU - F. Luis González Flecha
PY - 2026
DA - 2026/10/01
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27209233
UR - https://www.mdpi.com/1422-0067/27/20/9233
AB - Denaturation of small globular proteins is often described as a reversible two-state transition between native and denatured conformations. Conversely, multidomain and membrane proteins frequently populate several partially folded intermediate states during denaturation. Traditional thermodynamic analysis of fluorescence intensity often fails to reliably identify these intermediate states. Alternatively, spectral phasors provide an effective model-free way to monitor these complex conformational changes by resolving overlapping fluorescence signals with small differences in the position or shape of the spectra. Nevertheless, converting phasor trajectories into quantitative thermodynamic information remains challenging when quantum yields do not remain constant during a given process, thus limiting their application to protein stability studies. In this work, we introduce a thermodynamic framework for quantifying protein stability from phasor trajectories. We validate this formalism using simulated spectral data generated from two- and three-state transitions, including changes in fluorescence quantum yields, successfully recovering the thermodynamic parameters used to generate the simulations. Additionally, we applied this approach to analyze published data on urea-induced unfolding of E. coli Outer Membrane Protein A. Phasor analysis identified a two-state transition, and the thermodynamic parameters agreed well with those obtained from total fluorescence intensity, supporting the validity of the proposed approach. Finally, applying this framework to the SDS-induced denaturation of Archaeoglobus fulgidus Cu(I)-transport ATPase enabled a complete thermodynamic characterization of the three transitions identified by phasor analysis of intrinsic fluorescence, which could not be resolved using fluorescence intensity alone. This formalism establishes fluorescence spectral phasors as a powerful quantitative approach for protein stability analysis.
SN - 1422-0067
ER -