Investigation of α-Synuclein Fibril Structure by Site-directed Spin Labeling

Chen, Min; Margittai, Martin; Chen, Jeannie; Langen, Ralf

doi:10.1074/jbc.m700368200

Cited by 223 publications

(282 citation statements)

References 69 publications

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“…As discussed above, spectra of this type are indicative of extensive spin exchange, implying close proximity of nitroxide radicals. Consistent with this interpretation (26,27), generation of mixed fibrils containing 50% unlabeled protein resulted in a loss of signal amplitude because interspin distances increase and spectra become affected by dipolar broadening (Fig. 1B).…”

Section: Resultssupporting

confidence: 67%

“…Typically, spin-spin interactions between nitroxide labels in proteins are dominated by dipolar effects that result in a broadening of EPR spectra, providing distance information in the range of Ϸ8-25 Å (23,24). A second type of interaction, spin exchange, occurs at much smaller distances, when on the EPR time scale (Ͻ10 Ϫ7 s) multiple nitroxide labels are in sufficient proximity to allow orbital overlap (25)(26)(27). This type of interaction strikingly reduces classical three-line EPR spectra to a single feature.…”

Section: Resultsmentioning

confidence: 99%

“…This type of interaction strikingly reduces classical three-line EPR spectra to a single feature. Regardless of proximity, the presence of just two labels is insufficient to result in a single-line EPR spectrum (26)(27)(28), making these signals extremely unusual in proteins. However, spectra such as these have recently been reported for spinlabeled fibrils formed by tau (26) and ␣-synuclein (27).…”

Section: Resultsmentioning

confidence: 99%

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Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Cobb

Sönnichsen

Mchaourab³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

306

342

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Transmissible spongiform encephalopathies (TSEs) represent a group of fatal neurodegenerative diseases that are associated with conformational conversion of the normally monomeric and ␣-helical prion protein, PrP C , to the ␤-sheet-rich PrP Sc . This latter conformer is believed to constitute the main component of the infectious TSE agent. In contrast to high-resolution data for the PrP C monomer, structures of the pathogenic PrP Sc or synthetic PrP Sc -like aggregates remain elusive. Here we have used sitedirected spin labeling and EPR spectroscopy to probe the molecular architecture of the recombinant PrP amyloid, a misfolded form recently reported to induce transmissible disease in mice overexpressing an N-terminally truncated form of PrP C . Our data show that, in contrast to earlier, largely theoretical models, the conformational conversion of PrP C involves major refolding of the C-terminal ␣-helical region. The core of the amyloid maps to C-terminal residues from Ϸ160 -220, and these residues form single-molecule layers that stack on top of one another with parallel, in-register alignment of ␤-strands. This structural insight has important implications for understanding the molecular basis of prion propagation, as well as hereditary prion diseases, most of which are associated with point mutations in the region found to undergo a refolding to ␤-structure.EPR ͉ spin labeling ͉ transmissible spongiform encephalopathy

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Section: Resultssupporting

confidence: 67%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Cobb

Sönnichsen

Mchaourab³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

306

342

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“…Combining modern electron paramagnetic resonance (EPR) technologies and molecular biology methods to obtain recombinant proteins with specific sites for spin labeling provides powerful approaches to study molecular structures and interactions when either high-resolution NMR or X-ray techniques are difficult. Site-directed spin labeling EPR methods have been applied to study protein structures and protein-protein interactions in protein complexes (1), membrane proteins (2-4), amyloid peptides and fibrils (5)(6). Typically, a recombinant protein with a cysteine residue at a designated position is labeled with a sulfhydryl reactive spin label molecule, and EPR spectra of the labeled protein are obtained under different conditions.…”

mentioning

confidence: 99%

“…11 & 12) for spin labeling by (1-oxy-2,2,5,5-tetramethyl-3-pyrrolinyl-3-methyl) methanethiosulfonate (MTSSL), a widely used label (4,6,7). This labeling is reversible, and the label can be released with the addition of DTT (13).…”

mentioning

confidence: 99%

Potential artifacts in using a glutathione S-transferase fusion protein system and spin labeling electron paramagnetic resonance methods to study protein–protein interactions

Antoniou

Fung

2008

Analytical Biochemistry

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Site directed spin labeling EPR methods have been an important tool in studying protein-protein interactions. Labels are often attached to a cysteine residue side chain and spectra are acquired with and without binding partner(s) to provide information on the binding. This requires a knowledge of the location of the label, which is simplified if the label remains faithfully attached to the designated residue in the complex. We report a system where this is not the case because the label was extracted by dialysis-resistant glutathione molecules. Once this artifact is identified, spectral subtraction provides a simple solution for meaningful data interpretation.Protein-protein interactions are crucial in almost all biological processes such as immune responses, cell signaling and regulation (1). Molecular level studies to identify the site(s) of interaction and to determine structures for mechanistic insights toward association affinity are key approaches in understanding these processes (1). Combining modern electron paramagnetic resonance (EPR) technologies and molecular biology methods to obtain recombinant proteins with specific sites for spin labeling provides powerful approaches to study molecular structures and interactions when either high-resolution NMR or X-ray techniques are difficult. Site-directed spin labeling EPR methods have been applied to study protein structures and protein-protein interactions in protein complexes (1), membrane proteins (2-4), amyloid peptides and fibrils (5-6). Typically, a recombinant protein with a cysteine residue at a designated position is labeled with a sulfhydryl reactive spin label molecule, and EPR spectra of the labeled protein are obtained under different conditions. For protein-protein interaction studies, the spectra are obtained in the absence and presence of non-labeled binding partner(s). The chemical environments and/or the dynamics of the bound spin label without and with the binding partners are compared. This experimental process is often repeated with the cysteine residue at different positions in the protein, a method sometimes called site directed spin labeling EPR (7). In some cases, a qualitative inspection of spectra of labeled proteins with different binding partners is sufficient to provide molecular information, as in allosteric connections involved in the activation process of G proteins (8) or in investigating local conformational changes of Band 3 protein around a residue whose mutation causes hemolytic disease (9). Otherwise, spectral parameters that reflect label mobility or environment are obtained quantitatively from the spectra (7,8,10). Either qualitative or quantitative interpretation of these spectra requires a knowledge of the location of the label, which is simplified if the label remains faithfully attached to the designated cysteine residue upon interaction with binding partner(s). However, if the location of the bound spin label changes *To whom correspondence should be addressed; email: lfung@uic.edu. Publisher's Disclaimer...

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α‐Synuclein phase separation and amyloid aggregation are modulated by C‐terminal truncations

Huang

Wang

et al. 2022

FEBS Letters

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The aggregation of α‐synuclein (α‐Syn) is a key pathological hallmark of Parkinson's disease (PD). α‐Syn undergoes liquid‐liquid phase separation (LLPS) to drive amyloid aggregation. How the LLPS of α‐Syn is regulated remains largely unknown. Here, we discovered that the C‐terminal region modulates α‐Syn phase separation through electrostatic interactions. The wild‐type (WT) and PD disease‐related truncated α‐Syn can co‐exist in the condensates. The truncated α‐Syn could dramatically promote WT α‐Syn phase separation. Further studies demonstrated that the truncated α‐Syn accelerated WT α‐Syn turning to amyloid aggregates by modulation of phase separation. Together, our findings disclose the role of the C‐terminal domain in the LLPS of α‐Syn and pave the path for understanding the mechanism of truncated α‐Syn in PD pathology.

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Investigation of α-Synuclein Fibril Structure by Site-directed Spin Labeling

Cited by 223 publications

References 69 publications

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Molecular architecture of human prion protein amyloid: A parallel, in-register β-structure

Potential artifacts in using a glutathione S-transferase fusion protein system and spin labeling electron paramagnetic resonance methods to study protein–protein interactions

α‐Synuclein phase separation and amyloid aggregation are modulated by C‐terminal truncations

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