Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Shewmaker, Frank; Wickner, Reed B.; Tycko, Robert

doi:10.1073/pnas.0609638103

Cited by 272 publications

(334 citation statements)

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“…With single-site labeling, the dipolar recoupling data represent measurements of intermolecular distances, which can readily distinguish parallel from antiparallel β-sheets and establish the precise registry of backbone hydrogen bonds within the β-sheets. We have previously used 13 C fpRFDR-CT measurements to investigate β-sheet structures in fibrils formed by β-amyloid peptides (17,19,21) and by yeast prion proteins (18,63). Figure 10 compares experimental 13 C fpRFDR-CT data for AMY04 fibrils with simulations for ideal linear chains of 13 C nuclei with various internuclear spacings.…”

Section: Evidence For In-register Parallel β-Sheetsmentioning

confidence: 99%

“…Measurements of 13 C-13 C and 15 N-15 N nuclear magnetic dipole-dipole couplings (which are proportional to the inverse cube of internuclear distances) were carried out with the constanttime fpRFDR (fpRFDR-CT) technique, as previously described (54) and applied to other amyloid-forming peptides and proteins (17,18,21,63), using 20 kHz MAS frequencies and 15 μs fpRFDR π pulses. In 15 N-15 N distance measurements, designed to probe the peptide backbone conformation at sites where uniformly labeled residues occurred sequentially, spin polarization was transferred from backbone 15 N nuclei to directly bonded 13 C α nuclei for signal detection using a transferred echo double resonance (TEDOR) sequence (64,65) after the 15 N fpRFDR-CT period, as previously described (66).…”

Section: Solid State Nmrmentioning

confidence: 99%

“…One-dimensional (1D) RAD measurements were also performed on AMY10 fibrils to confirm the 2D results. In the 1D RAD measurements, a frequency-selective 13 C π/2 pulse following cross-polarization was used to prepare longitudinal spin polarization on the Phe15 aromatic carbons, which could then be transferred to other 13 C-labeled sites during the exchange period prior to signal detection after a hard π/2 pulse.Measurements of 13 C-13 C and 15 N-15 N nuclear magnetic dipole-dipole couplings (which are proportional to the inverse cube of internuclear distances) were carried out with the constanttime fpRFDR (fpRFDR-CT) technique, as previously described (54) and applied to other amyloid-forming peptides and proteins (17,18,21,63), using 20 kHz MAS frequencies and 15 μs fpRFDR π pulses. In 15 N-15 N distance measurements, designed to probe the peptide backbone conformation at sites where uniformly labeled residues occurred sequentially, spin polarization was transferred from backbone 15 N nuclei to directly bonded 13 C α nuclei for signal detection using a transferred echo double resonance (TEDOR) sequence (64,65) after the 15 N fpRFDR-CT period, as previously described (66).…”

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Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

et al. 2007

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The 37-residue amylin peptide, also known as islet amyloid polypeptide, forms fibrils that are the main peptide or protein component of amyloid that develops in the pancreas of type 2 diabetes patients. Amylin also readily forms amyloid fibrils in vitro that are highly polymorphic under typical experimental conditions. We describe a protocol for the preparation of synthetic amylin fibrils that exhibit a single predominant morphology, which we call a striated ribbon, in electron microscope and atomic force microscope images. Solid state nuclear magnetic resonance (NMR) measurements on a series of isotopically labeled samples indicate a single molecular structure within the striated ribbons. We use scanning transmission electron microscopy and several types of one-dimensional and two-dimensional solid state NMR techniques to obtain constraints on the peptide conformation and supramolecular structure in these amylin fibrils, and derive molecular structural models that are consistent with the experimental data. The basic structural unit in amylin striated ribbons, which we call the protofilament, contains four-layers of parallel β-sheets, formed by two symmetric layers of amylin molecules. The molecular structure of amylin protofilaments in striated ribbons closely resembles the protofilament in amyloid fibrils with similar morphology formed by the 40-residue β-amyloid peptide that is associated with Alzheimer's disease. Keywordsislet amyloid polypeptide; type 2 diabetes; amyloid fibril structure; electron microscopy; atomic force microscopy Amylin, also called islet amyloid polypeptide or IAPP, is a 37-residue peptide that is cosecreted with insulin by the β-cells of pancreas. The normal biological effects of amylin secretion include inhibition of glucagon secretion and reduction of the rate of gastric emptying, effects that contribute to the maintenance of postprandial glucose homeostasis (1). Amylin is the major peptide or protein component of the islet amyloid found in the pancreas of approximately 90% of type 2 (or non-insulin-dependent) diabetes patients (2,3). Fibrillar amylin has been shown * Corresponding author: Dr. Robert Tycko, National Institutes of Health, Building 5, Room 112, Bethesda, MD 20892-0520. phone 301-402-8272. fax 301-496-0825. e-mail robertty@mail.nih.gov.Supporting Information Available HPLC chromatograms from the purification of both reduced and oxidized amylin by reverse-phase chromatography ( Figure S1); FPLC chromatograms from the purification of both human and rodent amylin by size-exclusion chromatography ( Figure S2); 1D 13 C spectra of amylin fibrils with various isotopic labeling patterns, in lyophilized and rehydrated conditions ( Figures S3-S5); Table of 13 C NMR chemical shifts in amylin fibrils, TALOS predictions of backbone torsion angles based on these shifts, and torsion angles determined from 15 N fpRFDR-CT measurements (Table S1). This material is freely available on the World Wide Web at http://www.acs.org. to be toxic to cultured β-cells (4), and has been propo...

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Section: Evidence For In-register Parallel β-Sheetsmentioning

confidence: 99%

Section: Solid State Nmrmentioning

confidence: 99%

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Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

et al. 2007

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“…Using solid-state NMR, amyloids of the Alzheimer's A␤ peptide and the diabetes-associated amyloid peptide amylin are inregister parallel ␤-sheets (identical residues of different molecules aligned along the fiber long axis) (20)(21)(22) (23)(24)(25). Electron spin resonance studies showed in-register parallel ␤-sheet structures of amylin (26), ␣-synuclein (Parkinson's disease) (27), and Tau (Alzheimer's disease and other 'tauopathies') (28).…”

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The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

McGlinchey¹,

Shewmaker²,

McPhie³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Pmel17 is a melanocyte protein necessary for eumelanin deposition 1 in mammals and found in melanosomes in a filamentous form. The luminal part of human Pmel17 includes a region (RPT) with 10 copies of a partial repeat sequence, pt.e.gttp.qv., known to be essential in vivo for filament formation. We show that this RPT region readily forms amyloid in vitro, but only under the mildly acidic conditions typical of the lysosome-like melanosome lumen, and the filaments quickly become soluble at neutral pH. Under the same mildly acidic conditions, the Pmel filaments promote eumelanin formation. Electron diffraction, circular dichroism, and solidstate NMR studies of Pmel17 filaments show that the structure is rich in beta sheet. We suggest that RPT is the amyloid core domain of the Pmel17 filaments so critical for melanin formation.

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“…34). Solid-state NMR studies of amyloids of the prion domains of Sup35 (35) and Ure2p (36) as well as Ure2p 10 -39 (37) have shown that each is a parallel, in-register ␤-sheet structure, meaning that, for example, residue 35 of one molecule is aligned opposite residue 35 of the preceding molecule and the subsequent molecule in the fibril.…”

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Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure

Wickner

Dyda²,

Tycko³

2008

Proc. Natl. Acad. Sci. U.S.A.

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The [PIN ؉ ] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI ؉ ] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1⌬ strains have no known phenotype except for inability to propagate the [PIN ؉ ] prion. We used solidstate NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153-405) labeled with Tyr-1-13 C (14 residues), Leu-1-13 C (7 residues), or Ala-3-13 C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in ␤-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were Ϸ0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel ␤-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3-13 C indicate that the ␤-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN ؉ ]'s defining characteristic.T hree genetic criteria designed to identify infectious proteins [PIN ϩ ] has all of the genetic properties of a prion (4, 5), and was shown to be a self-propagating amyloid form of Rnq1p (4, 6), a protein rich in asparagine (N) and glutamine (Q), which had been shown to form self-propagating aggregates in vivo (7). Deletion of the RNQ1 gene produces no evident phenotype (7) (14), it is likely that variants of [PIN ϩ ] are different amyloid structures, although the precise nature of such differences remains unknown for any system at present.Aggregates formed in vitro from Rnq1p are filamentous and stain with thioflavin T (7). Circular dichroism indicates that these aggregates are high in ␤-sheet content and, on exposure to proteinase K, show partial resistance (6). These studies indicate that the Rnq1p aggregates are amyloid fibers. Moreover, these fibers formed in vitro from recombinant Rnq1p are infectious for yeast spheroplasts, efficiently transmitting the [PIN ϩ ] gene (6).Because amyloid is neither soluble nor crystalline, neither solution NMR nor x-ray crystallography is suitable for elucidating its detailed structure, although the study of amyloid-like microcrystals of short oligopeptides has revealed details of packing and sidechain interactions that are relevant to amyloid structures (15,16). Electron spin resonance studies have been used to study amylin (IAPP) and ␣-synuclein (17, 18) as well as amyloid of PrP (19). Solid-state NMR has been particularly useful for the elucidation of the structure of amyloids (reviewed in ref. 20). Selective labeling of particular amino acid residues with magnetic nuclear isotopes 13 C and 15 N, combined with pulse sequences designed...

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Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Cited by 272 publications

References 62 publications

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure

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Amyloid of the prion domain of Sup35p has an in-register parallel β-sheet structure

Cited by 272 publications

References 62 publications

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

Peptide Conformation and Supramolecular Organization in Amylin Fibrils: Constraints from Solid-State NMR

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

Amyloid of Rnq1p, the basis of the [ PIN + ] prion, has a parallel in-register β-sheet structure

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Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure