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1.  Unlike Twins: An NMR Comparison of Two α-Synuclein Polymorphs Featuring Different Toxicity 
PLoS ONE  2014;9(3):e90659.
We structurally compare, using solid-state NMR, two different polymorphs of α-synuclein which, as established recently, display contrasting biochemical properties, toxicity, and tropism for cells. We show that both forms, which can each be produced as a pure polymorph, are greatly different in secondary structure. While β-sheets are the dominating secondary structure elements for both polymorphs, they are markedly divergent in terms of number of elements, as well as their distribution. We demonstrate that all identified β-sheets feature an in-register parallel stacking for both polymorphs. The two forms show a different molecular arrangement in the unit cell and distinct dynamic features, while sharing a highly flexible C-terminal domain. The use of reproducible, well-identified conditions for sample preparation and the recording of identical NMR experiments allows for a direct comparison of the results.
doi:10.1371/journal.pone.0090659
PMCID: PMC3944079  PMID: 24599158
2.  Structural and functional characterization of two alpha-synuclein strains 
Nature Communications  2013;4:2575.
α-synuclein aggregation is implicated in a variety of diseases including Parkinson’s disease, dementia with Lewy bodies, pure autonomic failure and multiple system atrophy. The association of protein aggregates made of a single protein with a variety of clinical phenotypes has been explained for prion diseases by the existence of different strains that propagate through the infection pathway. Here we structurally and functionally characterize two polymorphs of α-synuclein. We present evidence that the two forms indeed fulfil the molecular criteria to be identified as two strains of α-synuclein. Specifically, we show that the two strains have different structures, levels of toxicity, and in vitro and in vivo seeding and propagation properties. Such strain differences may account for differences in disease progression in different individuals/cell types and/or types of synucleinopathies.
α-synuclein is implicated in neurodegenerative diseases. Bousset et al. generate two α-synuclein polymorphs and find differences in aggregation, function and toxicity, suggesting that these altered properties may be the cause for differences in disease progression.
doi:10.1038/ncomms3575
PMCID: PMC3826637  PMID: 24108358
3.  Prions 
Prion  2010;4(2):72-79.
The prion hypothesis1–3 states that the prion and non-prion form of a protein differ only in their 3D conformation and that different strains of a prion differ by their 3D structure.4,5 Recent technical developments have enabled solid-state NMR to address the atomic-resolution structures of full-length prions, and a first comparative study of two of them, HET-s and Ure2p, in fibrillar form, has recently appeared as a pair of companion papers.6,7 Interestingly, the two structures are rather different: HET-s features an exceedingly well-ordered prion domain and a partially disordered globular domain. Ure2p in contrast features a very well ordered globular domain with a conserved fold, and—most probably—a partially ordered prion domain.6 For HET-s, the structure of the prion domain is characterized at atomic-resolution. For Ure2p, structure determination is under way, but the highly resolved spectra clearly show that information at atomic resolution should be achievable.
PMCID: PMC2933054  PMID: 20424506
prion; NMR; solid-state NMR; MAS; structure; Ure2p; HET-s
4.  Mechanism of Inhibition of Enveloped Virus Membrane Fusion by the Antiviral Drug Arbidol 
PLoS ONE  2011;6(1):e15874.
The broad-spectrum antiviral arbidol (Arb) inhibits cell entry of enveloped viruses by blocking viral fusion with host cell membrane. To better understand Arb mechanism of action, we investigated its interactions with phospholipids and membrane peptides. We demonstrate that Arb associates with phospholipids in the micromolar range. NMR reveals that Arb interacts with the polar head-group of phospholipid at the membrane interface. Fluorescence studies of interactions between Arb and either tryptophan derivatives or membrane peptides reconstituted into liposomes show that Arb interacts with tryptophan in the micromolar range. Interestingly, apparent binding affinities between lipids and tryptophan residues are comparable with those of Arb IC50 of the hepatitis C virus (HCV) membrane fusion. Since tryptophan residues of membrane proteins are known to bind preferentially at the membrane interface, these data suggest that Arb could increase the strength of virus glycoprotein's interactions with the membrane, due to a dual binding mode involving aromatic residues and phospholipids. The resulting complexation would inhibit the expected viral glycoprotein conformational changes required during the fusion process. Our findings pave the way towards the design of new drugs exhibiting Arb-like interfacial membrane binding properties to inhibit early steps of virus entry, i.e., attractive targets to combat viral infection.
doi:10.1371/journal.pone.0015874
PMCID: PMC3026800  PMID: 21283579
5.  Kinetic analysis of protein aggregation monitored by real-time 2D solid-state NMR spectroscopy 
Journal of Biomolecular Nmr  2011;49(2):121-129.
It is shown that real-time 2D solid-state NMR can be used to obtain kinetic and structural information about the process of protein aggregation. In addition to the incorporation of kinetic information involving intermediate states, this approach can offer atom-specific resolution for all detectable species. The analysis was carried out using experimental data obtained during aggregation of the 10.4 kDa Crh protein, which has been shown to involve a partially unfolded intermediate state prior to aggregation. Based on a single real-time 2D 13C–13C transition spectrum, kinetic information about the refolding and aggregation step could be extracted. In addition, structural rearrangements associated with refolding are estimated and several different aggregation scenarios were compared to the experimental data.
doi:10.1007/s10858-011-9468-6
PMCID: PMC3042102  PMID: 21253842
Aggregation; Kinetic; Solid-state NMR; Real-time spectroscopy; Crh
6.  Simultaneous use of solution, solid-state NMR and X-ray crystallography to study the conformational landscape of the Crh protein during oligomerization and crystallization 
We explore, using the Crh protein dimer as a model, how information from solution NMR, solid-state NMR and X-ray crystallography can be combined using structural bioinformatics methods, in order to get insights into the transition from solution to crystal. Using solid-state NMR chemical shifts, we filtered intra-monomer NMR distance restraints in order to keep only the restraints valid in the solid state. These filtered restraints were added to solid-state NMR restraints recorded on the dimer state to sample the conformational landscape explored during the oligomerization process. The use of non-crystallographic symmetries then permitted the extraction of converged conformers subsets. Ensembles of NMR and crystallographic conformers calculated independently display similar variability in monomer orientation, which supports a funnel shape for the conformational space explored during the solution-crystal transition. Insights into alternative conformations possibly sampled during oligomerization were obtained by analyzing the relative orientation of the two monomers, according to the restraint precision. Molecular dynamics simulations of Crh confirmed the tendencies observed in NMR conformers, as a paradoxical increase of the distance between the two β1a strands, when the structure gets closer to the crystallographic structure, and the role of water bridges in this context.
PMCID: PMC3170007  PMID: 21918624
structural bioinformatics; NMR structure calculation; ARIA; non-crystallographic symmetry; crystallographic ensemble refinement; molecular dynamics simulation
7.  Structural constraints for the Crh protein from solid-state NMR experiments 
Journal of Biomolecular Nmr  2008;40(4):239-250.
We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-008-9229-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-008-9229-3
PMCID: PMC2579321  PMID: 18320329
Catabolite repression histidine-containing phosphocarrier protein (Crh); Distance constraints; MAS; 3D Protein structure; Solid-state NMR spectroscopy

Results 1-7 (7)