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Site-specific interaction between α-synuclein and membranes probed by NMR-observed methionine oxidation rates.

Maltsev AS, Chen J, Levine RL, Bax A - J. Am. Chem. Soc. (2013)

Bottom Line: The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein.The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa.When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
α-Synuclein (αS) is an intrinsically disordered protein that is water-soluble but also can bind negatively charged lipid membranes while adopting an α-helical conformation. Membrane affinity is increased by post-translational N-terminal acetylation, a common modification in all eukaryotic cells. In the presence of lipid vesicles containing a small fraction of peroxidized lipids, the N-terminal Met residues in αS (Met1 and Met5) rapidly oxidize while reducing the toxic lipid hydroperoxide to a nonreactive lipid hydroxide, whereas C-terminal Met residues remain unaffected. Met oxidation can be probed conveniently and quantitatively by NMR spectroscopy. The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein. The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa. When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.

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Overlays of the 800 MHz 1H–15N HSQCspectra of N-acetylated αS recorded after removal of lipid,showing the cross-peaks for residue L8 for samples harvested at differenttimes [30 min (black), 2 h (green), 8 h (blue), and 16 h (red)] afterincubation of the protein with a 5% w/v lipid mixture [100 μMαS, 150 mM NaCl, 20 mM sodium phosphate (pH 6), 15% DOPS, 25%DOPE, and 60% DOPC with 10 ± 0.5% peroxidization of the oleylchains] at 20 °C. The black spectrum is shown in its originalposition, and the others are displaced in 0.2 ppm steps in the 15N dimension for clarity. Oxidation state labels: NN, neitherM1 nor M5 oxidized; ON, only M1 oxidized; NO, only M5 oxidized; OO,both M1 and M5 oxidized.
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fig1: Overlays of the 800 MHz 1H–15N HSQCspectra of N-acetylated αS recorded after removal of lipid,showing the cross-peaks for residue L8 for samples harvested at differenttimes [30 min (black), 2 h (green), 8 h (blue), and 16 h (red)] afterincubation of the protein with a 5% w/v lipid mixture [100 μMαS, 150 mM NaCl, 20 mM sodium phosphate (pH 6), 15% DOPS, 25%DOPE, and 60% DOPC with 10 ± 0.5% peroxidization of the oleylchains] at 20 °C. The black spectrum is shown in its originalposition, and the others are displaced in 0.2 ppm steps in the 15N dimension for clarity. Oxidation state labels: NN, neitherM1 nor M5 oxidized; ON, only M1 oxidized; NO, only M5 oxidized; OO,both M1 and M5 oxidized.

Mentions: Figure 1 showsthe 1H–15N HSQC resonances of residueL8 for four samples harvestedat different times after excess lipid (q = 630) inthe form of SUVs (5% w/v) was added to a freshly prepared sample ofN-terminally acetylated αS. L8 falls in a particularly wellresolved region of the HSQC spectrum, and four resonances are observedfor it: the nonoxidized form of the protein (labeled “NN”)gives the most-upfield L8 resonance, and those corresponding to proteinwith oxidized M1 and natural M5 (“ON”), natural M1 andoxidized M5 (“NO”), and both M1 and M5 oxidized (“OO”)are increasingly shifted downfield.


Site-specific interaction between α-synuclein and membranes probed by NMR-observed methionine oxidation rates.

Maltsev AS, Chen J, Levine RL, Bax A - J. Am. Chem. Soc. (2013)

Overlays of the 800 MHz 1H–15N HSQCspectra of N-acetylated αS recorded after removal of lipid,showing the cross-peaks for residue L8 for samples harvested at differenttimes [30 min (black), 2 h (green), 8 h (blue), and 16 h (red)] afterincubation of the protein with a 5% w/v lipid mixture [100 μMαS, 150 mM NaCl, 20 mM sodium phosphate (pH 6), 15% DOPS, 25%DOPE, and 60% DOPC with 10 ± 0.5% peroxidization of the oleylchains] at 20 °C. The black spectrum is shown in its originalposition, and the others are displaced in 0.2 ppm steps in the 15N dimension for clarity. Oxidation state labels: NN, neitherM1 nor M5 oxidized; ON, only M1 oxidized; NO, only M5 oxidized; OO,both M1 and M5 oxidized.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585462&req=5

fig1: Overlays of the 800 MHz 1H–15N HSQCspectra of N-acetylated αS recorded after removal of lipid,showing the cross-peaks for residue L8 for samples harvested at differenttimes [30 min (black), 2 h (green), 8 h (blue), and 16 h (red)] afterincubation of the protein with a 5% w/v lipid mixture [100 μMαS, 150 mM NaCl, 20 mM sodium phosphate (pH 6), 15% DOPS, 25%DOPE, and 60% DOPC with 10 ± 0.5% peroxidization of the oleylchains] at 20 °C. The black spectrum is shown in its originalposition, and the others are displaced in 0.2 ppm steps in the 15N dimension for clarity. Oxidation state labels: NN, neitherM1 nor M5 oxidized; ON, only M1 oxidized; NO, only M5 oxidized; OO,both M1 and M5 oxidized.
Mentions: Figure 1 showsthe 1H–15N HSQC resonances of residueL8 for four samples harvestedat different times after excess lipid (q = 630) inthe form of SUVs (5% w/v) was added to a freshly prepared sample ofN-terminally acetylated αS. L8 falls in a particularly wellresolved region of the HSQC spectrum, and four resonances are observedfor it: the nonoxidized form of the protein (labeled “NN”)gives the most-upfield L8 resonance, and those corresponding to proteinwith oxidized M1 and natural M5 (“ON”), natural M1 andoxidized M5 (“NO”), and both M1 and M5 oxidized (“OO”)are increasingly shifted downfield.

Bottom Line: The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein.The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa.When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
α-Synuclein (αS) is an intrinsically disordered protein that is water-soluble but also can bind negatively charged lipid membranes while adopting an α-helical conformation. Membrane affinity is increased by post-translational N-terminal acetylation, a common modification in all eukaryotic cells. In the presence of lipid vesicles containing a small fraction of peroxidized lipids, the N-terminal Met residues in αS (Met1 and Met5) rapidly oxidize while reducing the toxic lipid hydroperoxide to a nonreactive lipid hydroxide, whereas C-terminal Met residues remain unaffected. Met oxidation can be probed conveniently and quantitatively by NMR spectroscopy. The results show that oxidation of Met1 reduces the rate of oxidation of Met5 and vice versa as a result of decreased membrane affinity of the partially oxidized protein. The effect of Met oxidation on the αS-membrane affinity extends over large distances, as in the V49M mutant, oxidation of Met1 and Met5 strongly impacts the oxidation rate of Met49 and vice versa. When not bound to membrane, oxidized Met1 and Met5 of αS are excellent substrates for methionine sulfoxide reductase (Msr), thereby providing an efficient vehicle for water-soluble Msr enzymes to protect the membrane against oxidative damage.

Show MeSH
Related in: MedlinePlus