Category Archives: Amyloids

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States #DNPNMR

Uluca, Boran, Thibault Viennet, Dušan Petrović, Hamed Shaykhalishahi, Franziska Weirich, Ayşenur Gönülalan, Birgit Strodel, Manuel Etzkorn, Wolfgang Hoyer, and Henrike Heise. “DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States.” Biophysical Journal 114, no. 7 (April 2018): 1614–23.

https://doi.org/10.1016/j.bpj.2018.02.011.

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein a-synuclein (a-syn), which plays a key role in the etiology of Parkinson’s disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called ‘‘random coil conformation’’ consists of a-helical and b-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar a-syn as well as in a-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.

Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy #DNPNMR

Nagaraj, M., et al., Surface Binding of TOTAPOL Assists Structural Investigations of Amyloid Fibrils by Dynamic Nuclear Polarization NMR Spectroscopy. Chembiochem, 2016. 17(14): p. 1308-11.

https://www.ncbi.nlm.nih.gov/pubmed/27147408

Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.

Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization

Potapov, A., et al., Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization. J Am Chem Soc, 2015. 137(25): p. 8294-307.

http://www.ncbi.nlm.nih.gov/pubmed/26068174

Self-assembly of amyloid-beta (Abeta) peptides in human brain tissue leads to neurodegeneration in Alzheimer’s disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Abeta self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Abeta (Abeta40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel beta-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.

Dynamic Nuclear Polarization NMR Spectroscopy: Revealing Multiple Conformations in Lipid-Anchored Peptide Vaccines

Koers, E.J., et al., Dynamic Nuclear Polarization NMR Spectroscopy: Revealing Multiple Conformations in Lipid-Anchored Peptide Vaccines. Angew Chem Int Ed Engl, 2013. 52(41): p. 10905-10908.

http://www.ncbi.nlm.nih.gov/pubmed/24039018

Sensitivity is the key: Dynamic nuclear polarization NMR spectroscopy provides structural information on liposomal vaccines targeting Alzheimer’s disease. DMPC/DMPG/Cholesterol mainly stabilizes extended structures of the lipid-anchored peptide, while in DMTAP/Cholesterol liposomes the peptide adopts a multitude of conformations including random-coil and extended structures.

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