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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.
Bhargava,
Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: a site-directed spin-labeling study.
2004, Pubmed
Bhargava,
Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: a site-directed spin-labeling study.
2004,
Pubmed
Bodner,
Multiple tight phospholipid-binding modes of alpha-synuclein revealed by solution NMR spectroscopy.
2009,
Pubmed
Bonifacino,
The mechanisms of vesicle budding and fusion.
2004,
Pubmed
Cattani,
Room-temperature in-cell EPR spectroscopy: alpha-Synuclein disease variants remain intrinsically disordered in the cell.
2017,
Pubmed
,
Xenbase
Das,
Membrane interactions of intrinsically disordered proteins: The example of alpha-synuclein.
2019,
Pubmed
Davidson,
Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes.
1998,
Pubmed
Dedmon,
Mapping long-range interactions in alpha-synuclein using spin-label NMR and ensemble molecular dynamics simulations.
2005,
Pubmed
Dikiy,
Semisynthetic and in Vitro Phosphorylation of Alpha-Synuclein at Y39 Promotes Functional Partly Helical Membrane-Bound States Resembling Those Induced by PD Mutations.
2016,
Pubmed
Drescher,
Spin-label EPR on alpha-synuclein reveals differences in the membrane binding affinity of the two antiparallel helices.
2008,
Pubmed
Eliezer,
Conformational properties of alpha-synuclein in its free and lipid-associated states.
2001,
Pubmed
Feix,
Conformational Changes and Membrane Interaction of the Bacterial Phospholipase, ExoU: Characterization by Site-Directed Spin Labeling.
2019,
Pubmed
Fusco,
Direct observation of the three regions in α-synuclein that determine its membrane-bound behaviour.
2014,
Pubmed
Gophane,
A semi-rigid isoindoline-derived nitroxide spin label for RNA.
2018,
Pubmed
Gordon-Grossman,
A combined pulse EPR and Monte Carlo simulation study provides molecular insight on peptide-membrane interactions.
2009,
Pubmed
Hubbell,
Identifying conformational changes with site-directed spin labeling.
2000,
Pubmed
Hänsel,
Evaluation of parameters critical for observing nucleic acids inside living Xenopus laevis oocytes by in-cell NMR spectroscopy.
2009,
Pubmed
,
Xenbase
Igarashi,
Distance determination in proteins inside Xenopus laevis oocytes by double electron-electron resonance experiments.
2010,
Pubmed
,
Xenbase
Izmailov,
Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories.
2020,
Pubmed
Jagtap,
Sterically shielded spin labels for in-cell EPR spectroscopy: analysis of stability in reducing environment.
2015,
Pubmed
Jakes,
Identification of two distinct synucleins from human brain.
1994,
Pubmed
Jao,
Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.
2008,
Pubmed
Klug,
Methods and applications of site-directed spin labeling EPR spectroscopy.
2008,
Pubmed
Lawless,
ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1.
2018,
Pubmed
Lomasney,
Phosphatidylinositol 4,5-bisphosphate binding to the pleckstrin homology domain of phospholipase C-delta1 enhances enzyme activity.
1996,
Pubmed
Maltsev,
Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties.
2012,
Pubmed
Manning,
AKT/PKB signaling: navigating downstream.
2007,
Pubmed
Maroteaux,
Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal.
1988,
Pubmed
Masaracchia,
Membrane binding, internalization, and sorting of alpha-synuclein in the cell.
2018,
Pubmed
Mitchell,
X-band rapid-scan EPR of nitroxyl radicals.
2012,
Pubmed
Qi,
Gd(III)-PyMTA label is suitable for in-cell EPR.
2014,
Pubmed
,
Xenbase
Receveur-Bréchot,
Assessing protein disorder and induced folding.
2006,
Pubmed
Robotta,
Alpha-Synuclein Disease Mutations Are Structurally Defective and Locally Affect Membrane Binding.
2017,
Pubmed
Robotta,
Alpha-synuclein binds to the inner membrane of mitochondria in an α-helical conformation.
2014,
Pubmed
Robotta,
Direct evidence of coexisting horseshoe and extended helix conformations of membrane-bound alpha-synuclein.
2011,
Pubmed
Robotta,
Locally resolved membrane binding affinity of the N-terminus of α-synuclein.
2012,
Pubmed
Sakai,
In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes.
2006,
Pubmed
,
Xenbase
Shvadchak,
The mode of α-synuclein binding to membranes depends on lipid composition and lipid to protein ratio.
2011,
Pubmed
Sikora,
Allosteric Signaling Is Bidirectional in an Outer-Membrane Transport Protein.
2016,
Pubmed
Stoll,
EasySpin, a comprehensive software package for spectral simulation and analysis in EPR.
2006,
Pubmed
Tao,
Annexin B12 Trimer Formation is Governed by a Network of Protein-Protein and Protein-Lipid Interactions.
2020,
Pubmed
Theillet,
Structural disorder of monomeric α-synuclein persists in mammalian cells.
2016,
Pubmed
Todd,
ESR spectra reflect local and global mobility in a short spin-labeled peptide throughout the alpha-helix----coil transition.
1991,
Pubmed
Ulmer,
Comparison of structure and dynamics of micelle-bound human alpha-synuclein and Parkinson disease variants.
2005,
Pubmed
