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Copper(II) binding to alpha-synuclein, the Parkinson's protein.

Lee JC, Gray HB, Winkler JR - J. Am. Chem. Soc. (2008)

Bottom Line: Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease.Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7.Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8013, USA. leej4@mail.nih.gov

ABSTRACT
Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

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Primary amino acid sequence of human α-synuclein. Potential side-chain copper(II) ligands (N-terminus, Lys, His, Asp, Glu, and Met) are colored. Underlined residues indicate the location of Trp probes.
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fig1: Primary amino acid sequence of human α-synuclein. Potential side-chain copper(II) ligands (N-terminus, Lys, His, Asp, Glu, and Met) are colored. Underlined residues indicate the location of Trp probes.

Mentions: We have employed tryptophan fluorescence measurements to probe Cu(II)−protein binding in four mutants containing single W substitutions (F4W, Y39W, F94W, and Y125W: Figure 1).(22) Upon addition of Cu(II) (0.3−60 μΜ), the tryptophan fluorescence is quenched site-specifically (Figures 1S and 2S in Supporting Information). The fluorophore at position 4 is the most strongly quenched (>80% at [Cu(II)] = [protein] = 5 µM, vide infra), while W39 exhibits minimal quenching until more than 1 equiv of Cu(II) has been added (∼20% fluorescence decrease at 10 equiv of Cu(II), Figure 3S in Supporting Information). Even though the participation of the N-terminal amino group(23) and residues in the vicinity of 3−9(24) have been implicated in Cu(II) binding, our observation was somewhat unexpected because H50 was identified previously as the Cu(II) ligand with the strongest affinity.23,24 Furthermore, neither W94 nor W125 reporter in the highly acidic (15 carboxylate groups) C-terminal region(25) is sensitive to the presence of Cu(II) under these solution conditions, and fluorescence energy transfer kinetics measured for W39→Y(NO2) pairs near H50 (Y19−W39 and W39−Y55) show no detectable changes upon the addition of Cu(II). The data confirm that Cu(II) binding to α-syn does not induce a significant conformational change between residues 19 and 55 (data not shown).


Copper(II) binding to alpha-synuclein, the Parkinson's protein.

Lee JC, Gray HB, Winkler JR - J. Am. Chem. Soc. (2008)

Primary amino acid sequence of human α-synuclein. Potential side-chain copper(II) ligands (N-terminus, Lys, His, Asp, Glu, and Met) are colored. Underlined residues indicate the location of Trp probes.
© Copyright Policy - open-access - ccc-price
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664836&req=5

fig1: Primary amino acid sequence of human α-synuclein. Potential side-chain copper(II) ligands (N-terminus, Lys, His, Asp, Glu, and Met) are colored. Underlined residues indicate the location of Trp probes.
Mentions: We have employed tryptophan fluorescence measurements to probe Cu(II)−protein binding in four mutants containing single W substitutions (F4W, Y39W, F94W, and Y125W: Figure 1).(22) Upon addition of Cu(II) (0.3−60 μΜ), the tryptophan fluorescence is quenched site-specifically (Figures 1S and 2S in Supporting Information). The fluorophore at position 4 is the most strongly quenched (>80% at [Cu(II)] = [protein] = 5 µM, vide infra), while W39 exhibits minimal quenching until more than 1 equiv of Cu(II) has been added (∼20% fluorescence decrease at 10 equiv of Cu(II), Figure 3S in Supporting Information). Even though the participation of the N-terminal amino group(23) and residues in the vicinity of 3−9(24) have been implicated in Cu(II) binding, our observation was somewhat unexpected because H50 was identified previously as the Cu(II) ligand with the strongest affinity.23,24 Furthermore, neither W94 nor W125 reporter in the highly acidic (15 carboxylate groups) C-terminal region(25) is sensitive to the presence of Cu(II) under these solution conditions, and fluorescence energy transfer kinetics measured for W39→Y(NO2) pairs near H50 (Y19−W39 and W39−Y55) show no detectable changes upon the addition of Cu(II). The data confirm that Cu(II) binding to α-syn does not induce a significant conformational change between residues 19 and 55 (data not shown).

Bottom Line: Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease.Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7.Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-8013, USA. leej4@mail.nih.gov

ABSTRACT
Variations in tryptophan fluorescence intensities confirm that copper(II) interacts with alpha-synuclein, a protein implicated in Parkinson's disease. Trp4 fluorescence decay kinetics measured for the F4W protein show that Cu(II) binds tightly (Kd 100 nM) near the N-terminus at pH 7. Work on a F4W/H50S mutant indicates that a histidine imidazole is not a ligand in this high-affinity site.

Show MeSH
Related in: MedlinePlus