Dataset Information

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils.

ABSTRACT: A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insight into the protein backbone conformation and dynamics in fibrils formed by (13)C,(15)N-labeled huPrP23-144. Surprisingly, we find that signals from approximately 100 residues (i.e., approximately 80% of the sequence) are not detected above approximately -20 degrees C in conventional solid-state NMR spectra. Sequential resonance assignments revealed that signals, which are observed, arise exclusively from residues in the region 112-141. These resonances are remarkably narrow, exhibiting average (13)C and (15)N linewidths of approximately 0.6 and 1 ppm, respectively. Altogether, the present findings indicate the existence of a compact, highly ordered core of huPrP23-144 amyloid encompassing residues 112-141. Analysis of (13)C secondary chemical shifts identified likely beta-strand segments within this core region, including beta-strand 130-139 containing critical residues 138 and 139. In contrast to this relatively rigid, beta-sheet-rich amyloid core, the remaining residues in huPrP23-144 amyloid fibrils under physiologically relevant conditions are largely unordered, displaying significant conformational dynamics.

SUBMITTER: Helmus JJ

PROVIDER: S-EPMC2359773 | biostudies-literature | 2008 Apr

REPOSITORIES: biostudies-literature

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Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils.

Helmus Jonathan J JJ Surewicz Krystyna K Nadaud Philippe S PS Surewicz Witold K WK Jaroniec Christopher P CP

Proceedings of the National Academy of Sciences of the United States of America 20080424 17

A C-terminally truncated Y145Stop variant of the human prion protein (huPrP23-144) is associated with a hereditary amyloid disease known as PrP cerebral amyloid angiopathy. Previous studies have shown that recombinant huPrP23-144 can be efficiently converted in vitro to the fibrillar amyloid state, and that residues 138 and 139 play a critical role in the amyloidogenic properties of this protein. Here, we have used magic-angle spinning solid-state NMR spectroscopy to provide high-resolution insi ...[more]

PMID: 18436646

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Project description:Amyloid aggregates of a C-truncated Y145Stop mutant of human prion protein, huPrP23-144, associated with a heritable amyloid angiopathy, have previously been shown to contain a compact, relatively rigid, and beta-sheet-rich approximately 30-residue amyloid core near the C-terminus under physiologically relevant conditions. In contrast, the remaining huPrP23-144 residues display considerable conformational dynamics, as evidenced by the absence of corresponding signals in cross-polarization (CP)-based solid-state NMR (SSNMR) spectra under ambient conditions and their emergence in analogous spectra recorded at low temperature on frozen fibril samples. Here, we present the direct observation of residues comprising the flexible N-terminal domain of huPrP23-144 amyloid by using 2D J-coupling-based magic-angle spinning (MAS) SSNMR techniques. Chemical shifts for these residues indicate that the N-terminal domain is effectively an ensemble of protein chains with random-coil-like conformations. Interestingly, a detailed analysis of signal intensities in CP-based 3D SSNMR spectra suggests that non-negligible molecular motions may also be occurring on the NMR time scale within the relatively rigid core of huPrP23-144 amyloid. To further investigate this hypothesis, quantitative measurements of backbone dipolar order parameters and transverse spin relaxation rates were performed for the core residues. The observed order parameters indicate that, on the submicrosecond time scale, these residues are effectively rigid and experience only highly restricted and relatively uniform motions similar to those characteristic for well-structured regions of microcrystalline proteins. On the other hand, significant variations in magnitude of transverse spin relaxation rates were noted for residues present at different locations within the core region and correlated with observed differences in spectral intensities. While interpreted only qualitatively at the present time, the extent of the observed variations in transverse relaxation rates is consistent with the presence of relatively slow, microsecond-millisecond time scale chemical exchange type phenomena within the huPrP23-144 amyloid core.

Project description:Many peptides and proteins self-assemble into amyloid fibrils. Examples include mammalian and fungal prion proteins, polypeptides associated with human amyloid diseases, and proteins that may have biologically functional amyloid states. To understand the propensity for polypeptides to form amyloid fibrils and to facilitate rational design of amyloid inhibitors and imaging agents, it is necessary to elucidate the molecular structures of these fibrils. Although fibril structures were largely mysterious 15 years ago, a considerable body of reliable structural information about amyloid fibril structures now exists, with essential contributions from solid state nuclear magnetic resonance (NMR) measurements. This Account reviews results from our laboratories and discusses several structural issues that have been controversial. In many cases, the amino acid sequences of amyloid fibrils do not uniquely determine their molecular structures. Self-propagating, molecular-level polymorphism complicates the structure determination problem and can lead to apparent disagreements between results from different laboratories, particularly when different laboratories study different polymorphs. For 40-residue β-amyloid (Aβ₁₋₄₀) fibrils associated with Alzheimer's disease, we have developed detailed structural models from solid state NMR and electron microscopy data for two polymorphs. These polymorphs have similar peptide conformations, identical in-register parallel β-sheet organizations, but different overall symmetry. Other polymorphs have also been partially characterized by solid state NMR and appear to have similar structures. In contrast, cryo-electron microscopy studies that use significantly different fibril growth conditions have identified structures that appear (at low resolution) to be different from those examined by solid state NMR. Based on solid state NMR and electron paramagnetic resonance (EPR) measurements, the in-register parallel β-sheet organization found in β-amyloid fibrils also occurs in many other fibril-forming systems. We attribute this common structural motif to the stabilization of amyloid structures by intermolecular interactions among like amino acids, including hydrophobic interactions and polar zippers. Surprisingly, we have recently identified and characterized antiparallel β-sheets in certain fibrils that are formed by the D23N mutant of Aβ₁₋₄₀, a mutant that is associated with early-onset, familial neurodegenerative disease. Antiparallel D23N-Aβ₁₋₄₀ fibrils are metastable with respect to parallel structures and, therefore, represent an off-pathway intermediate in the amyloid fibril formation process. Other methods have recently produced additional evidence for antiparallel β-sheets in other amyloid-formation intermediates. As an alternative to simple parallel and antiparallel β-sheet structures, researchers have proposed β-helical structural models for some fibrils, especially those formed by mammalian and fungal prion proteins. Solid state NMR and EPR data show that fibrils formed in vitro by recombinant PrP have in-register parallel β-sheet structures. However, the structure of infectious PrP aggregates is not yet known. The fungal HET-s prion protein has been shown to contain a β-helical structure. However, all yeast prions studied by solid state NMR (Sup35p, Ure2p, and Rnq1p) have in-register parallel β-sheet structures, with their Gln- and Asn-rich N-terminal segments forming the fibril core.

Dataset Information

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils.

Publications

Molecular conformation and dynamics of the Y145Stop variant of human prion protein in amyloid fibrils.

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