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

https://doi.org/10.1016/j.bbagen.2020.129770Get rights and content
Under a Creative Commons license
open access

Highlights

  • Reports a novel approach for analysis of FRET.

  • Uses polarized multidimensional fluorescence measurements.

  • Resolves overlapped donor and acceptor spectra using chemometrics.

  • Enables calculation of more accurate quenching and energy transfer parameters.

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

Fluorescence
Förster resonance energy transfer
Anisotropy
Protein
Chemometrics
Modelling

Abbreviations

ANS, 1
8-anilinonaphathalene sulfonate
ARMES
anisotropy resolved multidimensional emission spectroscopy
EEM
excitation emission matrices
FRET
Förster resonance energy transfer
HH
horizontal-horizontal
HSA
Human Serum Albumin
HV
horizontal-vertical
IFE
inner filter effect
LOR
limit of reporting
MCR
multivariate curve resolution
MDF
multi-dimensional fluorescence
PARAFAC
parallel factor
pEEM
polarized EEM
pTSFS
polarized TSFS
Trp
tryptophan
TSFS
total synchronous fluorescent scans
Tyr
tyrosine
VV
vertical-vertical
VH
vertical-horizontal

Cited by (0)