Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance. Smith, A., N., Märker, K., Piretra, T., Boatz, J., C., Matlahov, I., Kodali, R., Hediger, S., van der Wel, P., C., A., & De Paëpe, G. Journal of the American Chemical Society, 140(44):14576-14580, UTC, 11, 2018.
Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance [link]Website  abstract   bibtex   
A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13 C− 13 C distance constraints. P rotein aggregates that are the hallmark of many incurable protein-misfolding disorders continue to be challenging targets for structural studies. However, knowledge of their structures is essential to understand the molecular mechanism of protein misfolding and aggregation. 1 Magic angle spinning (MAS) ssNMR has provided not only high-resolution structures of protein fibrils but also unique and crucial insights into the atomic-level underpinnings of polymorphic aggregated states. 2,3 The latter studies directly compare 1D and 2D spectra of distinct fiber polymorphs, as "spectroscopic fingerprints", taking advantage of the structural sensitivity of NMR chemical shifts. Thus far, these experiments rely on multidimensional correlation spectroscopy applied to poly-peptides with 13 C/ 15 N isotope enrichment, which limits or prevents applications to samples that are hard or impossible to label, such as patient-or animal-derived materials. Here, an approach for determining high-resolution structural fingerprints of protein aggregates at NA is presented and shows how the absence of isotopic enrichment is a substantial advantage for these kinds of structure-based analyses. This approach is demonstrated on neurotoxic aggregates formed by the first exon of mutant huntingtin protein (with 44 Gln residues; Q44-HttEx1) and a peptide-based model of its polyQ core (D 2 Q 15 K 2), both at NA (Figure 1a). Notably, we report for the first time the extraction of long-range 13 C− 13 C distances on protein fibrils at NA, yielding intermolecular constraints that map out the core arrangement of Q44-HttEx1 fibrils. These measurements are enabled both by the dilute network of NA 13 C spins (1.1%) and by enhancing sensitivity with dynamic nuclear polarization (DNP). PolyQ expansion diseases, such as HD, are caused by an autosomal dominant genetic mutation that leads to an expanded CAG trinucleotide repeat in affected genes. 4 Figure 1. (a) Secondary structure schematic of Q44-HttEx1 and D 2 Q 15 K 2. (b) 13 C spectra of Q44-HttEx1 with and without $μ$w irradiation. The polyQ and PPII-helix resonances are labeled. Communication pubs.acs.org/JACS Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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 title = {Structural Fingerprinting of Protein Aggregates by Dynamic Nuclear Polarization-Enhanced Solid-State NMR at Natural Isotopic Abundance},
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 year = {2018},
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 abstract = {A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13 C− 13 C distance constraints. P rotein aggregates that are the hallmark of many incurable protein-misfolding disorders continue to be challenging targets for structural studies. However, knowledge of their structures is essential to understand the molecular mechanism of protein misfolding and aggregation. 1 Magic angle spinning (MAS) ssNMR has provided not only high-resolution structures of protein fibrils but also unique and crucial insights into the atomic-level underpinnings of polymorphic aggregated states. 2,3 The latter studies directly compare 1D and 2D spectra of distinct fiber polymorphs, as "spectroscopic fingerprints", taking advantage of the structural sensitivity of NMR chemical shifts. Thus far, these experiments rely on multidimensional correlation spectroscopy applied to poly-peptides with 13 C/ 15 N isotope enrichment, which limits or prevents applications to samples that are hard or impossible to label, such as patient-or animal-derived materials. Here, an approach for determining high-resolution structural fingerprints of protein aggregates at NA is presented and shows how the absence of isotopic enrichment is a substantial advantage for these kinds of structure-based analyses. This approach is demonstrated on neurotoxic aggregates formed by the first exon of mutant huntingtin protein (with 44 Gln residues; Q44-HttEx1) and a peptide-based model of its polyQ core (D 2 Q 15 K 2), both at NA (Figure 1a). Notably, we report for the first time the extraction of long-range 13 C− 13 C distances on protein fibrils at NA, yielding intermolecular constraints that map out the core arrangement of Q44-HttEx1 fibrils. These measurements are enabled both by the dilute network of NA 13 C spins (1.1%) and by enhancing sensitivity with dynamic nuclear polarization (DNP). PolyQ expansion diseases, such as HD, are caused by an autosomal dominant genetic mutation that leads to an expanded CAG trinucleotide repeat in affected genes. 4 Figure 1. (a) Secondary structure schematic of Q44-HttEx1 and D 2 Q 15 K 2. (b) 13 C spectra of Q44-HttEx1 with and without $μ$w irradiation. The polyQ and PPII-helix resonances are labeled. Communication pubs.acs.org/JACS Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX},
 bibtype = {article},
 author = {Smith, Adam N. and Märker, Katharina and Piretra, Talia and Boatz, Jennifer C. and Matlahov, Irina and Kodali, Ravindra and Hediger, Sabine and van der Wel, Patrick C. A. and De Paëpe, Gaël},
 journal = {Journal of the American Chemical Society},
 number = {44}
}

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