58 research outputs found

    Structural characterization of supramolecular assemblies by 13C spin dilution and 3D solid-state NMR.

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    13C spin diluted protein samples can be produced using [1-13C] and [2-13C]-glucose (Glc) carbon sources in the bacterial growth medium. The 13C spin dilution results in favorable 13C spectral resolution and polarization transfer behavior. We recently reported the combined use of [1-13C]- and [2-13C]-Glc labeling to facilitate the structural analysis of insoluble and non-crystalline biological systems by solid-state NMR (ssNMR), including sequential assignment, detection of long-range contacts and structure determination of macromolecular assemblies. In solution NMR the beneficial properties of sparsely labeled samples using [2-13C]-glycerol (13C labeled Cα sites on a 12C diluted background) have recently been exploited to provide a bi-directional assignment method (Takeuchi et al. in J Biomol NMR 49(1):17–26, 2011 ). Inspired by this approach and our own recent results using [2-13C]-Glc as carbon sources for the simplification of ssNMR spectra, we present a strategy for a bi-directional sequential assignment of solid-state NMR resonances and additionally the detection of long-range contacts using the combination of 13C spin dilution and 3D NMR spectroscopy. We illustrate our results with the sequential assignment and the collection of distance restraints on an insoluble and non-crystalline supramolecular assembly, the Salmonella typhimurium type III secretion system needle

    Atomic structure and handedness of the building block of a biological assembly.

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    Noncovalent supramolecular assemblies possess in general several unique subunit subunit interfaces.The basic building block of such an assembly consists of several subunits and contains all unique interfaces. Atomic-resolution structures of monomeric subunits are typically accessed by crystallography or solution NMR and fitted into electron microscopy density maps. However, the structure of the intact building block in the assembled state remains unknown with this hybrid approach. Here, we present the solid-state NMR atomic structure of the building block of the type III secretion system needle. The building block structure consists of a homotetrameric subunit complex with three unique supramolecular interfaces. Side-chain positions at the interfaces were solved at atomic detail. The high-resolution structure reveals unambiguously the helical handedness of the assembly, determined to be right-handed for the type III secretion system needle.Additionally, the axial rise per subunit could be extracted from the tetramer structure and independently validated by mass-per-length measurements

    Structural and molecular basis of cross-seeding barriers in amyloids

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    Neurodegenerative disorders are frequently associated with beta-sheet-rich amyloid deposits. Amyloid-forming proteins can aggregate under different structural conformations known as strains, which can exhibit a prion-like behavior and distinct pathophenotypes. Precise molecular determinants defining strain specificity and cross-strain interactions (cross-seeding) are currently unknown. The HET-s prion protein from the fungus Podospora anserina represents a model system to study the fundamental properties of prion amyloids. Here, we report the amyloid prion structure of HELLF, a distant homolog of the model prion HET-s. We find that these two amyloids, sharing only 17% sequence identity, have nearly identical beta-solenoid folds but lack cross-seeding ability in vivo, indicating that prion specificity can differ in extremely similar amyloid folds. We engineer the HELLF sequence to explore the limits of the sequence-to-fold conservation and to pinpoint determinants of cross-seeding and prion specificity. We find that amyloid fold conservation occurs even at an exceedingly low level of identity to HET-s (5%). Next, we derive a HELLF-based sequence, termed HEC, able to breach the cross-seeding barrier in vivo between HELLF and HET-s, unveiling determinants controlling cross-seeding at residue level. These findings show that virtually identical amyloid backbone structures might not be sufficient for cross-seeding and that critical side-chain positions could determine the seeding specificity of an amyloid fold. Our work redefines the conceptual boundaries of prion strain and sheds light on key molecular features concerning an important class of pathogenic agents

    Structural investigations of molecular machines by solid-state NMR.

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    Essential biological processes such as cell motion, signaling,protein synthesis, and pathogen-host interactions rely on multifunctional molecular machines containing supramolecular assemblies, that is, noncovalently assembled protein subunits. Scientists would like to acquire a detailed atomic view of the complete molecular machine to understand its assembly process and functions. Structural biologists have used various approaches to obtain structural information such as X-ray crystallography, solution NMR, and electron microscopy. The inherent insolubility and large size of these multicomponent assemblies restrict the use of solution NMR, and their noncrystallinity and elongated shapes present obstacles to X-ray crystallography studies. Not limited by molecular weight or crystallinity, solid-state NMR (ssNMR) allows for structural investigations of supramolecular assemblies such as helical filaments, cross-β fibrils, or membrane-embedded oligomeric proteins. In this Account, we describe recent progress in the application of ssNMR to the elucidation of atomic structures of supramolecular assemblies. We highlight ssNMR methods to determine the subunit interfaces in symmetric arrangements. Our use of [1-(13)C]- or [2-(13)C]-glucose as a carbon source during bacterial protein expression results in significant (13)C spin dilution that drastically improves the spectral quality and enables us to detect meaningful structural restraints. Moreover, we can unequivocally determine intermolecular restraints using mixed [(1:1)1-(13)C/2-(13)C]-glucose labeled assemblies. We recently illustrated the power of this methodology with the structure determination of the type III secretion system (T3SS) needle. One crucial aspect in elucidating the atomic structure of these large multicomponent complexes is to determine the subunit-subunit interfaces. Notably, we could probe the needle subunit interfaces by collecting (13)C-(13)C intermolecular restraints. In contrast, these interfaces are not accessible via high-resolution cryo-EM. This approach is readily applicable to other supramolecular assemblies containing symmetrically repeating protein subunits, and could be combined with other techniques to get a more complete picture of multicomponent structures. To determine near-atomic structures of assemblies of biological interest, researchers could combine ssNMR data collected at the subunit interfaces with the envelope obtained from cryo-EM and potentially with monomeric subunit crystal structures

    Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins.

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    Proton-detected solid-state NMR was applied to a highly deuterated insoluble, non-crystalline biological assembly, the Salmonella typhimurium type iii secretion system (T3SS) needle. Spectra of very high resolution and sensitivity were obtained at a low protonation level of 10-20% at exchangeable amide positions. We developed efficient experimental protocols for resonance assignment tailored for this system and the employed experimental conditions. Using exclusively dipolar-based interspin magnetization transfers, we recorded two sets of 3D spectra allowing for an almost complete backbone resonance assignment of the needle subunit PrgI. The additional information provided by the well-resolved proton dimension revealed the presence of two sets of resonances in the N-terminal helix of PrgI, while in previous studies employing 13C detection only a single set of resonances was observed

    BSH-CP based 3D solid-state NMR experiments for protein resonance assignment.

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    We have recently presented band-selective homonuclear cross-polarization (BSH-CP) as an efficient method for CO-CA transfer in deuterated as well as protonated solid proteins. Here we show how the BSH-CP CO-CA transfer block can be incorporated in a set of three-dimensional (3D) solid-state NMR (ssNMR) pulse schemes tailored for resonance assignment of proteins at high static magnetic fields and moderate magic-angle spinning rates. Due to the achieved excellent transfer efficiency of 33 % for BSH-CP, a complete set of 3D spectra needed for unambiguous resonance assignment could be rapidly recorded within 1 week for the model protein ubiquitin. Thus we expect that BSH-CP could replace the typically used CO-CA transfer schemes in well-established 3D ssNMR approaches for resonance assignment of solid biomolecules
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