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1.  Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes 
The EMBO Journal  2012;32(2):204-218.
Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.
The phenylalanine-glycine (FG) domains of vertebrate nucleoporins assemble into hydrogels with different sieving characteristics for macromolecules. Nup98 forms the tightest filter, which is relieved by O-linked glycosylation.
doi:10.1038/emboj.2012.302
PMCID: PMC3553378  PMID: 23202855
exportin; FG hydrogel; importin; nuclear pore complex; O-glycosylation
2.  Solid-state NMR [13C,15N] resonance assignments of the nucleotide-binding domain of a bacterial cyclic nucleotide-gated channel 
Biomolecular Nmr Assignments  2012;6(2):225-229.
Channels regulated by cyclic nucleotides are key signalling proteins in several biological pathways. The regulatory aspect is conferred by a C-terminal cyclic nucleotide-binding domain (CNBD). We report resonance assignments of the CNBD of a bacterial mlCNG channel obtained using 2D and 3D solid-state NMR under Magic-angle Spinning conditions. A secondary chemical shift analysis of the 141 residue protein suggests a three-dimensional fold seen in earlier X-ray and solution-state NMR work and points to spectroscopic polymorphism for a selected set of resonances.
Electronic supplementary material
The online version of this article (doi:10.1007/s12104-012-9363-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s12104-012-9363-4
PMCID: PMC3438399  PMID: 22302441
Cyclic nucleotide-binding domain; Cyclic AMP; Solid-state NMR; Magic-angle Spinning
3.  Fractional deuteration applied to biomolecular solid-state NMR spectroscopy 
Journal of Biomolecular Nmr  2011;52(2):91-101.
Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and D2O medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at Hα and Hβ positions reduce spectral congestion in (1H,13C,15N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR.
Electronic supplementary material
The online version of this article (doi:10.1007/s10858-011-9585-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10858-011-9585-2
PMCID: PMC3277825  PMID: 22105305
Assignment; Deuteration; Ion channel; MAS; Solid-state NMR; Structural constraints
4.  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
5.  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
6.  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
7.  High-Resolution 3D Structure Determination of Kaliotoxin by Solid-State NMR Spectroscopy 
PLoS ONE  2008;3(6):e2359.
High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from 1H/1H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 Å and 1.3 Å for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.
doi:10.1371/journal.pone.0002359
PMCID: PMC2387072  PMID: 18523586

Results 1-7 (7)