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J Phys Chem Lett
2021 Mar 11;129:2471-2475. doi: 10.1021/acs.jpclett.0c03583.
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Intracellular Protein-Lipid Interactions Studied by Rapid-Scan Electron Paramagnetic Resonance Spectroscopy.
Braun TS
,
Stehle J
,
Kacprzak S
,
Carl P
,
Höfer P
,
Subramaniam V
,
Drescher M
.
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Protein-membrane interactions play key roles in essential cellular processes; studying these interactions in the cell is a challenging task of modern biophysical chemistry. A prominent example is the interaction of human α-synuclein (αS) with negatively charged membranes. It has been well-studied in vitro, but in spite of the huge amount of lipid membranes in the crowded environment of biological cells, to date, no interactions have been detected in cells. Here, we use rapid-scan (RS) electron paramagnetic resonance (EPR) spectroscopy to study αS interactions with negatively charged vesicles in vitro and upon transfection of the protein and lipid vesicles into model cells, i.e., oocytes of Xenopus laevis. We show that protein-vesicle interactions are reflected in RS spectra in vitro and in cells, which enables time-resolved monitoring of protein-membrane interaction upon transfection into cells. Our data suggest binding of a small fraction of αS to endogenous membranes.
Figure 1. Schematic drawing
of αS (pink) (a) in solution and (b) bound
to a lipid membrane (blue). M-proxyl label for RS EPR experiment is
represented by its structural formula (green; not to scale). (c) Simulated4 absorption EPR spectra of M-proxyl labeled αS
A27C based on spectral fitting of continuous wave (CW) EPR measurements27 for αS in the absence of lipids (dashed
line, pink) and in the presence of negatively charged lipid vesicles
(blue) and uncharged lipid vesicles (yellow, overlaying with the pink
spectrum).
Figure 2. Experimental RS EPR spectra in vitro. (a) RS EPR
of M-proxyl labeled αS A27C in the presence of negatively charged
POPG LUVs (blue) compared to the presence of POPC LUVs (yellow), or
in absence of vesicles (dashed line, pink). (b) Measurement of M-proxyl
labeled αS A27C Δ2–11 in the presence of POPG LUVs
(violet) compared to the absence of vesicles (dashed line, green).
All spectra were normalized to the spectral maximum.
Figure 3. RS EPR spectra of M-proxyl labeled αS A27C. (a)
Vesicle binding
was achieved by in vitro incubation of αS with
LUVs for 30 min prior to microinjection into oocytes. In-cell spectra
of αS in the presence of POPG LUVs (blue) or POPC LUVs (yellow)
compared to the in-cell spectrum upon microinjection of protein without
lipids (pink). (b) Lipid vesicles were microinjected into oocytes.
Thirty min later, αS was injected into the same cells. Spectrum
in the presence of POPG LUVs of the first 10 min interval of the measurement
(dashed line, dark blue, 7 min after injection) compared to the third
10 min interval (bright blue) and the in-cell spectrum of protein
alone (pink).
Figure 4. (a) Protein–lipid interaction factors
(ξ) for αS
in absence of LUVs (pink), or in the presence of POPG (blue) or POPC
(yellow) LUVs corresponding to the spectra shown in Figures 1–3 A. (b) Quantification of vesicle binding in the cell by calculation
of ξ in a time-resolved manner. Microinjection of αS A27C
in oocytes with addition of POPG LUVs (blue) compared to the absence
(pink) of artificial vesicles and microinjection of A27C Δ2–11
(green). Empirical fits (Table S3) were
applied as a guide to the eye.
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