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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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Ratios of 1H–15N HSQC peak heightsin the presence (I) and absence (I0) of lipid as functions of total lipid concentration.Results are shown for residue L8 in a partially oxidized sample ofN-terminally acetylated WT αS containing a mixture of all fourM1/M5 oxidation states: NN (blue), ON (red), NO (green), and OO (magenta).The αS sample initially was harvested 4 h after addition of5% w/v partially oxidized SUVs (see the Figure 1 caption), and after lipid removal, nonoxidized SUVs were added toyield samples containing 100 μM αS in 20 mM sodium phosphate(pH 6). The SUV lipid composition was 30% DOPS, 50% DOPE, and 20%DOPC.
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fig2: Ratios of 1H–15N HSQC peak heightsin the presence (I) and absence (I0) of lipid as functions of total lipid concentration.Results are shown for residue L8 in a partially oxidized sample ofN-terminally acetylated WT αS containing a mixture of all fourM1/M5 oxidation states: NN (blue), ON (red), NO (green), and OO (magenta).The αS sample initially was harvested 4 h after addition of5% w/v partially oxidized SUVs (see the Figure 1 caption), and after lipid removal, nonoxidized SUVs were added toyield samples containing 100 μM αS in 20 mM sodium phosphate(pH 6). The SUV lipid composition was 30% DOPS, 50% DOPE, and 20%DOPC.

Mentions: Upon addition of fresh SUVs to a lipid-free wild-type(WT) αSsample containing a mixture of oxidation states of its Met residues,the binding of each form to the lipid could be monitored simultaneouslyby the attenuation of their signal intensities. As noted previously,exchange between the free and lipid-bound states of αS occursin the slow-exchange limit, with no significant impact on the linewidths and positions.25 The four formsof the protein were impacted to dramatically different extents bythe presence of lipid (Figure 2). Completeobliteration of the N-terminal intensities was observed for the NNprotein upon addition of an 8-fold molar excess (800 μM totallipid, ∼0.08% w/v), whereas the OO species remained virtuallyunaffected up to 5 mM total lipid (∼0.5% w/v); the NO and ONspecies were attenuated at intermediate levels. The much smaller signalattenuations for NO and ON compared with NN are indicative of weakerbinding of these partially oxidized states to the lipid, requiringthe use of four different oxidation rate constants: kNN-ON, kNN-NO, kON-OO, and kNO-OO. Fitting the harvest-time dependence of theresonance intensities of the four αS forms (Figure 3A) to the respective rate laws (eq S1 in the SI) gave rate constant values that confirmedthe considerable decrease in the M1 (M5) oxidation rateafter oxidation of M5 (M1) (kNO-OO < kNN-ON; kON-OO < kNN-NO; Table 1).


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)

Ratios of 1H–15N HSQC peak heightsin the presence (I) and absence (I0) of lipid as functions of total lipid concentration.Results are shown for residue L8 in a partially oxidized sample ofN-terminally acetylated WT αS containing a mixture of all fourM1/M5 oxidation states: NN (blue), ON (red), NO (green), and OO (magenta).The αS sample initially was harvested 4 h after addition of5% w/v partially oxidized SUVs (see the Figure 1 caption), and after lipid removal, nonoxidized SUVs were added toyield samples containing 100 μM αS in 20 mM sodium phosphate(pH 6). The SUV lipid composition was 30% DOPS, 50% DOPE, and 20%DOPC.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585462&req=5

fig2: Ratios of 1H–15N HSQC peak heightsin the presence (I) and absence (I0) of lipid as functions of total lipid concentration.Results are shown for residue L8 in a partially oxidized sample ofN-terminally acetylated WT αS containing a mixture of all fourM1/M5 oxidation states: NN (blue), ON (red), NO (green), and OO (magenta).The αS sample initially was harvested 4 h after addition of5% w/v partially oxidized SUVs (see the Figure 1 caption), and after lipid removal, nonoxidized SUVs were added toyield samples containing 100 μM αS in 20 mM sodium phosphate(pH 6). The SUV lipid composition was 30% DOPS, 50% DOPE, and 20%DOPC.
Mentions: Upon addition of fresh SUVs to a lipid-free wild-type(WT) αSsample containing a mixture of oxidation states of its Met residues,the binding of each form to the lipid could be monitored simultaneouslyby the attenuation of their signal intensities. As noted previously,exchange between the free and lipid-bound states of αS occursin the slow-exchange limit, with no significant impact on the linewidths and positions.25 The four formsof the protein were impacted to dramatically different extents bythe presence of lipid (Figure 2). Completeobliteration of the N-terminal intensities was observed for the NNprotein upon addition of an 8-fold molar excess (800 μM totallipid, ∼0.08% w/v), whereas the OO species remained virtuallyunaffected up to 5 mM total lipid (∼0.5% w/v); the NO and ONspecies were attenuated at intermediate levels. The much smaller signalattenuations for NO and ON compared with NN are indicative of weakerbinding of these partially oxidized states to the lipid, requiringthe use of four different oxidation rate constants: kNN-ON, kNN-NO, kON-OO, and kNO-OO. Fitting the harvest-time dependence of theresonance intensities of the four αS forms (Figure 3A) to the respective rate laws (eq S1 in the SI) gave rate constant values that confirmedthe considerable decrease in the M1 (M5) oxidation rateafter oxidation of M5 (M1) (kNO-OO < kNN-ON; kON-OO < kNN-NO; Table 1).

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