Prion Amyloid Polymorphs - The Tag Might Change It All.

Sup35p is a protein from Saccharomyces cerevisiae. It can propagate using a prion-like mechanism, which means that it can recruit non-prion soluble Sup35p into insoluble fibrils. Sup35p is a large protein showing three distinct domains, N, M and an extended globular domain. We have previously studied the conformations of the full-length and truncated NM versions carrying poly-histidine tags on the N-terminus. Comparison with structural data from C-terminally poly-histidine tagged NM from the literature surprisingly revealed discrepancies. Here we investigated fibrils from the untagged, as well as a C-terminally poly-histidine tagged NM construct, using solid-state NMR. We find that the conformation of untagged NM is very close to the N-terminally tagged NM and confirms our previous findings. The C-terminal poly-histidine tag, in contrast, drastically changes the NM fibril structure, and yields data consistent with results obtained previously on this construct. We conclude that the C-terminally located Sup35p globular domain influences the structure of the fibrillar core at the N domain, as previously shown. We further conclude, based on the present data, that small tags on NM C-terminus have a substantial, despite different, impact. Modifications at this remote localization thus shows an unexpected influence on the fibril structure, and importantly also its propensity to induce [PSI+].

Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate.

Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13C and 15N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway.

Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR.

Conformational studies of membrane proteins remain a challenge in the field of structural biology, and in particular the investigation of the proteins in a native-like lipid environment. Solid-state NMR presents a valuable opportunity for this, and we present here three critical steps in the solid-state NMR sample preparation, i.e., membrane reconstitution of the protein in native lipids, rotor filling, and sample quality assessment, at the example of the Bacillus subtilis ATP-binding cassette transporter BmrA.

Monitoring ssDNA Binding to the DnaB Helicase from Helicobacter pylori by Solid-State NMR Spectroscopy.

DnaB helicases are bacterial, ATP-driven enzymes that unwind double-stranded DNA during DNA replication. Herein, we study the sequential binding of the "non-hydrolysable" ATP analogue AMP-PNP and of single-stranded (ss) DNA to the dodecameric DnaB helicase from Helicobacter pylori using solid-state NMR. Phosphorus cross-polarization experiments monitor the binding of AMP-PNP and DNA to the helicase. 13 C chemical-shift perturbations (CSPs) are used to detect conformational changes in the protein upon binding. The helicase switches upon AMP-PNP addition into a conformation apt for ssDNA binding, and AMP-PNP is hydrolyzed and released upon binding of ssDNA. Our study sheds light on the conformational changes which are triggered by the interaction with AMP-PNP and are needed for ssDNA binding of H. pylori DnaB in vitro. They also demonstrate the level of detail solid-state NMR can provide for the characterization of protein-DNA interactions and the interplay with ATP or its analogues.

Variability and conservation of structural domains in divide-and-conquer approaches.

The use of protein building blocks for the structure determination of multidomain proteins and protein-protein complexes, also known as the "divide and conquer" approach, is an important strategy for obtaining protein structures. Atomic-resolution X-ray or NMR data of the individual domains are combined with lower-resolution electron microscopy maps or X-ray data of the full-length protein or the protein complex. Doing so, it is often assumed that the individual domain structures remain invariant in the context of the superstructure. In this work, we show the potentials and limitations of NMR to validate this approach at the example of the dodecameric DnaB helicase from Helicobacter pylori. We investigate how sequentially assigned spectra, as well as unassigned spectral fingerprints can be used to indicate the conservation of individual domains, and also to highlight conformational differences.

Solid-state NMR chemical-shift perturbations indicate domain reorientation of the DnaG primase in the primosome of Helicobacter pylori.

We here investigate the interactions between the DnaB helicase and the C-terminal domain of the corresponding DnaG primase of Helicobacter pylori using solid-state NMR. The difficult crystallization of this 387 kDa complex, where the two proteins interact in a six to three ratio, is circumvented by simple co-sedimentation of the two proteins directly into the MAS-NMR rotor. While the amount of information that can be extracted from such a large protein is still limited, we can assign a number of amino-acid residues experiencing significant chemical-shift perturbations upon helicase-primase complex formation. The location of these residues is used as a guide to model the interaction interface between the two proteins in the complex. Chemical-shift perturbations also reveal changes at the interaction interfaces of the hexameric HpDnaB assembly on HpDnaG binding. A structural model of the complex that explains the experimental findings is obtained.

Solid-state NMR sequential assignments of the N-terminal domain of HpDnaB helicase.

We present solid-state NMR assignments of the N-terminal domain of the DnaB helicase from Helicobacter pylori (153 residues) in its microcrystalline form. We use a sequential resonance assignment strategy based on three-dimensional NMR experiments. The resonance assignments obtained are compared with automated resonance assignments computed with the ssFLYA algorithm. An analysis of the (13)C secondary chemical shifts determines the position of the secondary structure elements in this α-helical protein.

Efficient and stable reconstitution of the ABC transporter BmrA for solid-state NMR studies.

We present solid-state NMR sample preparation and first 2D spectra of the Bacillus subtilis ATP-binding cassette (ABC) transporter BmrA, a membrane protein involved in multidrug resistance. The homodimeric 130-kDa protein is a challenge for structural characterization due to its membrane-bound nature, size, inherent flexibility and insolubility. We show that reconstitution of this protein in lipids from Bacillus subtilis at a lipid-protein ratio of 0.5 w/w allows for optimal protein insertion in lipid membranes with respect to two central NMR requirements, high signal-to-noise in the spectra and sample stability over a time period of months. The obtained spectra point to a well-folded protein and a highly homogenous preparation, as witnessed by the narrow resonance lines and the signal dispersion typical for the expected secondary structure distribution of BmrA. This opens the way for studies of the different conformational states of the transporter in the export cycle, as well as on interactions with substrates, via chemical-shift fingerprints and sequential resonance assignments.

A sedimented sample of a 59†kDa dodecameric helicase yields high-resolution solid-state NMR spectra.

Crystal clear: Preparing solid-state NMR samples that yield high-resolution spectra displaying high sensitivity is time-consuming and complicated. A sample of the 59 kDa protein DnaB, prepared simply by preparative centrifugation, provides spectra that are as good as the ones from carefully grown microcrystals.

Prion fibrils of Ure2p assembled under physiological conditions contain highly ordered, natively folded modules.

The difference between the prion and the non-prion form of a protein is given solely by its three-dimensional structure, according to the prion hypothesis. It has been shown that solid-state NMR can unravel the atomic-resolution three-dimensional structure of prion fragments but, in the case of Ure2p, no highly resolved spectra are obtained from the isolated prion domain. Here, we demonstrate that the spectra of full-length fibrils of Ure2p interestingly lead to highly resolved solid-state NMR spectra. Prion fibrils formed under physiological conditions are therefore well-ordered objects on the molecular level. Comparing the full-length NMR spectra with the corresponding spectra of the prion and globular domains in isolation reveals that the globular part in particular shows almost perfect structural order. The NMR linewidths in these spectra are as narrow as the ones observed in crystals of the isolated globular domain. For the prion domain, the spectra reflect partial disorder, suggesting structural heterogeneity, both in isolation and in full-length Ure2p fibrils, although to different extents. The spectral quality is surprising in the light of existing structural models for Ure2p and in comparison to the corresponding spectra of the only other full-length prion fibrils (HET-s) investigated so far. This opens the exciting perspective of an atomic-resolution structure determination of the fibrillar form of a prion whose assembly is not accompanied by significant conformational changes and documents the structural diversity underlying prion propagation.

Characterization of different water pools in solid-state NMR protein samples.

We observed and characterized two distinct signals originating from different pools of water protons in solid-state NMR protein samples, namely from crystal water which exchanges polarization with the protein (on the NMR timescale) and is located in the protein-rich fraction at the periphery of the magic-angle spinning (MAS) sample container, and supernatant water located close to the axis of the sample container. The polarization transfer between the water and the protein can be probed by two-dimensional exchange spectroscopy, and we show that the supernatant water does not interact with protein on the timescale of the experiments. The two water pools have different spectroscopic properties, including resonance frequency, longitudinal, transverse and rotating frame relaxation times. The supernatant water can be removed almost completely physically or can be frozen selectively. Both measures lead to an enhancement of the quality factor of the probe circuit, accompanied by an improvement of the experimental signal/noise, and greatly simplify solvent-suppression by substantially reducing the water signal. We also present a tool, which allows filling solid-state NMR sample containers in a more efficient manner, greatly reducing the amount of supernatant water and maximizing signal/noise.

Methyl proton contacts obtained using heteronuclear through-bond transfers in solid-state NMR spectroscopy.

A two-dimensional proton-mediated carbon-carbon correlation experiment that relies on through-bond heteronuclear magnetization transfers is demonstrated in the context of solid-state NMR of proteins. This new experiment, dubbed J-CHHC by analogy to the previously developed dipolar CHHC techniques, is shown to provide selective and sensitive correlations in the methyl region of 2D spectra of crystalline organic compounds. The method is then demonstrated on a microcrystalline sample of the dimeric protein Crh (2 x 10.4 kDa). A total of 34 new proton-proton contacts involving side-chain methyl groups were observed in the J-CHHC spectrum, which had not been observed with the conventional experiment. The contacts were then used as additional distance restraints for the 3D structure determination of this microcrystalline protein. Upon addition of these new distance restraints, which are in large part located in the hydrophobic core of the protein, the root-mean-square deviation with respect to the X-ray structure of the backbone atom coordinates of the 10 best conformers of the new ensemble of structures is reduced from 1.8 to 1.1 A.

Structural constraints for the Crh protein from solid-state NMR experiments.

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 x 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.

3D structure determination of the Crh protein from highly ambiguous solid-state NMR restraints.

In a wide variety of proteins, insolubility presents a challenge to structural biology, as X-ray crystallography and liquid-state NMR are unsuitable. Indeed, no general approach is available as of today for studying the three-dimensional structures of membrane proteins and protein fibrils. We here demonstrate, at the example of the microcrystalline model protein Crh, how high-resolution 3D structures can be derived from magic-angle spinning solid-state NMR distance restraints for fully labeled protein samples. First, we show that proton-mediated rare-spin correlation spectra, as well as carbon-13 spin diffusion experiments, provide enough short, medium, and long-range structural restraints to obtain high-resolution structures of this 2 x 10.4 kDa dimeric protein. Nevertheless, the large number of 13C/15N spins present in this protein, combined with solid-state NMR line widths of about 0.5-1 ppm, induces substantial ambiguities in resonance assignments, preventing 3D structure determination by using distance restraints uniquely assigned on the basis of their chemical shifts. In the second part, we thus demonstrate that an automated iterative assignment algorithm implemented in a dedicated solid-state NMR version of the program ARIA permits to resolve the majority of ambiguities and to calculate a de novo 3D structure from highly ambiguous solid-state NMR data, using a unique fully labeled protein sample. We present, using distance restraints obtained through the iterative assignment process, as well as dihedral angle restraints predicted from chemical shifts, the 3D structure of the fully labeled Crh dimer refined at a root-mean-square deviation of 1.33 A.

Determining the geometry of strongly hydrogen-bonded silanols in a layered hydrous silicate by solid-state nuclear magnetic resonance.

High-resolution solid-state NMR spectroscopy is exploited to obtain structural constraints involving strongly hydrogen-bonded silanols in octosilicate, a prominent member of the layered hydrous sodium silicates. Proton-silicon cross-polarization dynamics reveals that octosilicate contains two types of Q(3) silicons present in hydrogen-bonded -Si-O-Hcdots, three dots, centeredO-Si- and -Si-O(-)-type sites which can only be distinguished by their different abilities to cross polarize and the different mobilities of neighboring hydrous species. The theoretical analysis of the oscillating components of the polarization transfer buildup curves suggests that the model of heteronuclear pairs is an adequate description of the quantum spin system within hydrogen-bonded -Si-O-Hcdots, three dots, centeredO-Si- fragments. We also show that dipolar modulated, slow speed magic-angle (29)Si NMR spectrum provides unique geometric information on strongly hydrogen-bonded silanols. The dipolar modulated spinning sidebands contain all the information necessary to determine the internuclear Sicdots, three dots, centeredH distances as well as the magnitude and orientation of the principal elements of the (29)Si chemical shielding tensor in the molecular frame. The data provide definite proof of the intralayer character of strongly hydrogen-bonded silanol groups in a bridging, albeit not symmetric, position between neighboring tetrahedra. The approach developed in this work may be useful to obtain structural information on related layered alkali metal silicates, silica gels as well as on other classes of microporous materials.

Straightforward detection of the secondary ionisation of the phosphate group and pK determinations by high-resolution solid-state 31P NMR.

The suitability of high-resolution solid-state 31P NMR for a straightforward determination of the protonation state of phosphate groups as well as of their pK2 values extracted from solid state mono : dianionic ratios has been demonstrated.

An experimental and theoretical study of the 13C and 31P chemical shielding tensors in solid O-phosphorylated amino acids.

A series of l and dl forms of O-phosphorylated amino acids (serine, threonine, tyrosine) have been studied by using solid-state multinuclear NMR spectroscopy and ab initio calculations. Principal elements of the (13)C and (31)P chemical shielding tensors have been measured and discussed in relation to zwitterionic structures and intermolecular contacts. DFT calculations have been compared with experimental data showing their ability to reproduce experimentally obtained tensor values in this challenging class of compounds. The changes of orientation of (31)P chemical shielding tensor with respect to the molecular frame in the presence of hydrogen bonds have been revealed and discussed on the ground of theoretical calculations. The measurements of internuclear P...P distances, based on Zeeman magnetization exchange between (31)P spins with differing chemical shielding tensor orientations, were exploited for a clear distinction between enantiomers and racemates.