Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)

Fiona Gordon; Saioa Elcoroaristizabal; Alan G Ryder

doi:10.1016/j.bbagen.2020.129770

Modelling Förster resonance energy transfer (FRET) using anisotropy resolved multi-dimensional emission spectroscopy (ARMES)

Biochim Biophys Acta Gen Subj. 2021 Feb;1865(2):129770. doi: 10.1016/j.bbagen.2020.129770. Epub 2020 Oct 22.

Authors

Fiona Gordon¹, Saioa Elcoroaristizabal², Alan G Ryder³

Affiliations

¹ Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland, Galway, Galway H91 CF50, Ireland. Electronic address: F.GORDON2@nuigalway.ie.
² Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland, Galway, Galway H91 CF50, Ireland. Electronic address: elcoroaristizabal@nuigalway.ie.
³ Nanoscale BioPhotonics Laboratory, School of Chemistry, National University of Ireland, Galway, Galway H91 CF50, Ireland. Electronic address: alan.ryder@nuigalway.ie.

PMID: 33214128
DOI: 10.1016/j.bbagen.2020.129770

Abstract

Background: Förster Resonance Energy Transfer (FRET) is widely used to study the structure and dynamics of biomolecular systems and also causes the non-linear fluorescence response observed in multi-fluorophore proteins. Accurate FRET analysis, in terms of measuring changes in donor and acceptor spectra and energy transfer efficiency is therefore critical.

Methods: We demonstrate a novel quantitative FRET analysis using anisotropy resolved multidimensional emission spectroscopy (ARMES) in a Human Serum Albumin (HSA) and 1,8-anilinonaphathalene sulfonate (ANS) model. ARMES combines 4D measurement of polarized excitation emission matrices (pEEM) with multivariate data analysis to spectrally resolve contributing fluorophores. Multivariate analysis (Parallel Factor, PARAFAC and restricted Tucker3) was used to resolve fluorophore contributions and for modelling the quenching of HSA emission and the HSA-ANS interactions.

Results: pEEM spectra were modelled using Tucker3 which accommodates non-linearities introduced by FRET and a priori chemical knowledge was used to optimise the solution, thus resolving three components: HSA emission, ANS emission from indirect FRET excitation, and ANS emission from direct excitation. Perpendicular emission measurements were more sensitive to indirectly excited acceptor emission. PARAFAC modelling of HSA, donor emission, separated ANS FRET interacting (Tryptophan) and non-interacting (Tyrosine) components. This enabled a new way of calculating quenching constants using the multi-dimensional emission of individual donor fluorophores.

Conclusions: FRET efficiency could be calculated using the multi-dimensional, resolved emission of the interacting donor fluorophores only which yielded higher ET efficiencies compared to conventional methods.

General significance: Shows the potential of multidimensional fluorescence measurements and data analysis for more accurate FRET modelling in proteins.

Keywords: Anisotropy; Chemometrics; Fluorescence; Förster resonance energy transfer; Modelling; Protein.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Algorithms
Anilino Naphthalenesulfonates / chemistry*
Anisotropy
Fluorescence Resonance Energy Transfer / methods
Fluorescent Dyes / chemistry*
Humans
Models, Molecular
Serum Albumin, Human / chemistry*

Substances

Anilino Naphthalenesulfonates
Fluorescent Dyes
1-anilino-8-naphthalenesulfonate
Serum Albumin, Human