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Robert Tycko - One of the best experts on this subject based on the ideXlab platform.
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the α helical c terminal domain of full length recombinant prp converts to an in register parallel β Sheet Structure in prp fibrils evidence from solid state nuclear magnetic resonance
Biochemistry, 2010Co-Authors: Robert Tycko, Regina Savtchenko, Valeriy G Ostapchenko, Natallia Makarava, Ilia V BaskakovAbstract:We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-Sheets in these fibrils have an in-register parallel Structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core. Although unique site-specific assignments of the solid state NMR signals cannot be obtained from these spectra, analysis with a Monte Carlo/simulated annealing algorithm suggests that the core is comprised primarily of residues in the 173−224 range. These results are consistent with earlier electron paramagnetic resonance studies of fibrils formed by residues 90−231 of the human PrP sequence, formed under somewhat different conditions [Cobb, N. J., Sonnichsen, F. D., McHaourab, H., and Surewicz, W. K. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 18946−18951], suggesting that an in-register parallel β-Sheet Structure formed by the C-terminal end may be a general feature of PrP fibrils prepared in vitro.
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the functional curli amyloid is not based on in register parallel β Sheet Structure
Journal of Biological Chemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Fred Dyda, Ryan P Mcglinchey, Kent R Thurber, Peter Mcphie, Reed B WicknerAbstract:The extracellular curli proteins of Enterobacteriaceae form fibrous Structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β Structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli Structure is not based on an in-register parallel β-Sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like Structure but are not sufficient to establish such a Structure definitively.
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two prion variants of sup35p have in register parallel β Sheet Structures independent of hydration
Biochemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Dmitry Kryndushkin, Bo Chen, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1α respectively. Different [PSI+] isolates (prion variants) may have distinct biological properties, associated with different amyloid Structures. Here we use solid state NMR to examine the Structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β-Sheet Structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-Sheet Structure. Transmission of the [PSI+] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-le...
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Amyloids of Shuffled Prion Domains That Form Prions Have a Parallel In-Register β-Sheet Structure†
Biochemistry, 2008Co-Authors: Frank Shewmaker, Eric D Ross, Robert Tycko, Reed B WicknerAbstract:The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are self-propagating amyloid forms of Ure2p and Sup35p, respectively. The Q/N-rich N-terminal domains of each protein are necessary and sufficient for the prion properties of these proteins, forming in each case their amyloid cores. Surprisingly, shuffling either prion domain, leaving amino acid content unchanged, does not abrogate the ability of the proteins to become prions. The discovery that the amino acid composition of a polypeptide, not the specific sequence order, determines prion capability seems contrary to the standard folding paradigm that amino acid sequence determines protein fold. The shuffleability of a prion domain further suggests that the β-Sheet Structure is of the parallel in-register type, and indeed, the normal Ure2 and Sup35 prion domains have such a Structure. We demonstrate that two shuffled Ure2 prion domains capable of being prions form parallel in-register β-Sheet Structures, and our data indicate the same conclusion for...
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characterization of β Sheet Structure in ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Reed B Wickner, Alasdair C Steven, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular Structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-Sheets in Ure2p1-89 fibrils have an in-register parallel Structure. An in-register parallel β-Sheet Structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
Reed B Wickner - One of the best experts on this subject based on the ideXlab platform.
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amyloid of the candida albicans ure2p prion domain is infectious and has an in register parallel β Sheet Structure
Biochemistry, 2011Co-Authors: Abbi L Engel, Frank Shewmaker, Herman K Edskes, Fred Dyda, Reed B WicknerAbstract:Ure2p of Candida albicans (Ure2albicans or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2glabrata) cannot, even though the Ure2glabrata N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2cerevisiae) than Ure2albicans is. We show that the N-terminal N/Q-rich prion domain of Ure2albicans forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p1–90, we show that this infectious amyloid has an in-register parallel β-Sheet Structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2glabrata does not readily form amyloid, and that formed upon prolonged incubation is not infectious.
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the functional curli amyloid is not based on in register parallel β Sheet Structure
Journal of Biological Chemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Fred Dyda, Ryan P Mcglinchey, Kent R Thurber, Peter Mcphie, Reed B WicknerAbstract:The extracellular curli proteins of Enterobacteriaceae form fibrous Structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β Structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli Structure is not based on an in-register parallel β-Sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like Structure but are not sufficient to establish such a Structure definitively.
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two prion variants of sup35p have in register parallel β Sheet Structures independent of hydration
Biochemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Dmitry Kryndushkin, Bo Chen, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1α respectively. Different [PSI+] isolates (prion variants) may have distinct biological properties, associated with different amyloid Structures. Here we use solid state NMR to examine the Structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β-Sheet Structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-Sheet Structure. Transmission of the [PSI+] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-le...
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Amyloids of Shuffled Prion Domains That Form Prions Have a Parallel In-Register β-Sheet Structure†
Biochemistry, 2008Co-Authors: Frank Shewmaker, Eric D Ross, Robert Tycko, Reed B WicknerAbstract:The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are self-propagating amyloid forms of Ure2p and Sup35p, respectively. The Q/N-rich N-terminal domains of each protein are necessary and sufficient for the prion properties of these proteins, forming in each case their amyloid cores. Surprisingly, shuffling either prion domain, leaving amino acid content unchanged, does not abrogate the ability of the proteins to become prions. The discovery that the amino acid composition of a polypeptide, not the specific sequence order, determines prion capability seems contrary to the standard folding paradigm that amino acid sequence determines protein fold. The shuffleability of a prion domain further suggests that the β-Sheet Structure is of the parallel in-register type, and indeed, the normal Ure2 and Sup35 prion domains have such a Structure. We demonstrate that two shuffled Ure2 prion domains capable of being prions form parallel in-register β-Sheet Structures, and our data indicate the same conclusion for...
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characterization of β Sheet Structure in ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Reed B Wickner, Alasdair C Steven, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular Structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-Sheets in Ure2p1-89 fibrils have an in-register parallel Structure. An in-register parallel β-Sheet Structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
Frank Shewmaker - One of the best experts on this subject based on the ideXlab platform.
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amyloid of the candida albicans ure2p prion domain is infectious and has an in register parallel β Sheet Structure
Biochemistry, 2011Co-Authors: Abbi L Engel, Frank Shewmaker, Herman K Edskes, Fred Dyda, Reed B WicknerAbstract:Ure2p of Candida albicans (Ure2albicans or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2glabrata) cannot, even though the Ure2glabrata N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2cerevisiae) than Ure2albicans is. We show that the N-terminal N/Q-rich prion domain of Ure2albicans forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p1–90, we show that this infectious amyloid has an in-register parallel β-Sheet Structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2glabrata does not readily form amyloid, and that formed upon prolonged incubation is not infectious.
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the functional curli amyloid is not based on in register parallel β Sheet Structure
Journal of Biological Chemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Fred Dyda, Ryan P Mcglinchey, Kent R Thurber, Peter Mcphie, Reed B WicknerAbstract:The extracellular curli proteins of Enterobacteriaceae form fibrous Structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β Structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli Structure is not based on an in-register parallel β-Sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like Structure but are not sufficient to establish such a Structure definitively.
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two prion variants of sup35p have in register parallel β Sheet Structures independent of hydration
Biochemistry, 2009Co-Authors: Frank Shewmaker, Robert Tycko, Dmitry Kryndushkin, Bo Chen, Reed B WicknerAbstract:The [PSI+] prion is a self-propagating amyloid of the Sup35 protein, normally a subunit of the translation termination factor, but impaired in this vital function when in the amyloid form. The Sup35 N, M, and C domains are the amino-terminal prion domain, a connecting polar domain, and the essential C-terminal domain resembling eukaryotic elongation factor 1α respectively. Different [PSI+] isolates (prion variants) may have distinct biological properties, associated with different amyloid Structures. Here we use solid state NMR to examine the Structure of infectious Sup35NM amyloid fibrils of two prion variants. We find that both variants have an in-register parallel β-Sheet Structure, both in the fully hydrated form and in the lyophilized form. Moreover, we confirm that some leucine residues in the M domain participate in the in-register parallel β-Sheet Structure. Transmission of the [PSI+] prion by amyloid fibrils of Sup35NM and transmission of the [URE3] prion by amyloid fibrils of recombinant full-le...
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Amyloids of Shuffled Prion Domains That Form Prions Have a Parallel In-Register β-Sheet Structure†
Biochemistry, 2008Co-Authors: Frank Shewmaker, Eric D Ross, Robert Tycko, Reed B WicknerAbstract:The [URE3] and [PSI+] prions of Saccharomyces cerevisiae are self-propagating amyloid forms of Ure2p and Sup35p, respectively. The Q/N-rich N-terminal domains of each protein are necessary and sufficient for the prion properties of these proteins, forming in each case their amyloid cores. Surprisingly, shuffling either prion domain, leaving amino acid content unchanged, does not abrogate the ability of the proteins to become prions. The discovery that the amino acid composition of a polypeptide, not the specific sequence order, determines prion capability seems contrary to the standard folding paradigm that amino acid sequence determines protein fold. The shuffleability of a prion domain further suggests that the β-Sheet Structure is of the parallel in-register type, and indeed, the normal Ure2 and Sup35 prion domains have such a Structure. We demonstrate that two shuffled Ure2 prion domains capable of being prions form parallel in-register β-Sheet Structures, and our data indicate the same conclusion for...
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amyloid of the prion domain of sup35p has an in register parallel β Sheet Structure
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Frank Shewmaker, Reed B Wickner, Robert TyckoAbstract:The [PSI+] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Sup35p, a subunit of the translation termination factor. Using solid-state NMR we have examined the Structure of amyloid fibrils formed in vitro from purified recombinant Sup351–253, consisting of the glutamine- and asparagine-rich N-terminal 123-residue prion domain (N) and the adjacent 130-residue highly charged M domain. Measurements of magnetic dipole–dipole couplings among 13C nuclei in a series of Sup35NM fibril samples, 13C-labeled at backbone carbonyl sites of Tyr, Leu, or Phe residues or at side-chain methyl sites of Ala residues, indicate intermolecular 13C–13C distances of ≈0.5 nm for nearly all sites in the N domain. Certain sites in the M domain also exhibit intermolecular distances of ≈0.5 nm. These results indicate that an in-register parallel β-Sheet Structure underlies the [PSI+] prion phenomenon.
Ulrich Baxa - One of the best experts on this subject based on the ideXlab platform.
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characterization of β Sheet Structure in ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Reed B Wickner, Alasdair C Steven, D E Anderson, L N Marekov, W M Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular Structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-Sheets in Ure2p1-89 fibrils have an in-register parallel Structure. An in-register parallel β-Sheet Structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
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characterization of beta Sheet Structure in ure2p1 89 yeast prion fibrils by solid state nuclear magnetic resonance
Biochemistry, 2007Co-Authors: Ulrich Baxa, Reed B Wickner, Alasdair C Steven, D E Anderson, L N Marekov, Waiming Yau, Robert TyckoAbstract:Residues 1−89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular Structure of amyloid fibrils formed by Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-Sheets in Ure2p1-89 fibrils have an in-register parallel Structure. An in-register parallel β-Sheet Structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1−89. Two-dimensional NMR spectra of uniformly labeled Ure2p1-89 fibrils, even when fully hydrated, show NMR line...
Yoshitaka Ishii - One of the best experts on this subject based on the ideXlab platform.
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constraints on supramolecular Structure in amyloid fibrils from two dimensional solid state nmr spectroscopy with uniform isotopic labeling
Journal of the American Chemical Society, 2003Co-Authors: Robert Tycko, Yoshitaka IshiiAbstract:We show that strong constraints on supramolecular Structure in amyloid fibrils can be obtained from solid-state nuclear magnetic resonance measurements on samples with uniformly 13C-labeled segments. The measurements exploit two-dimensional (2D) 13C-13C exchange spectroscopy in conjunction with high-speed magic angle spinning, with proton-mediated exchange of 13C nuclear spin magnetization as recently demonstrated by Baldus and co-workers (J. Am. Chem. Soc. 2002, 124, 9704-9705). Proton-mediated 2D exchange spectra of fibrils formed by residues 16-22 of the 40-residue Alzheimer's beta-amyloid peptide show strong nonsequential, intermolecular cross-peaks between alpha-carbons that dictate an antiparallel beta-Sheet Structure in which residue 16+k aligns with residue 22-k. The strong alpha/alpha cross-peaks are absent from conventional, direct 2D exchange spectra. Proton-mediated 2D exchange spectra of fibrils formed by residues 11-25 indicate an antiparallel beta-Sheet Structure with a pH-dependent intermolecular alignment. In contrast, proton-mediated 2D exchange spectra of fibrils formed by the full-length beta-amyloid peptide are consistent with a parallel beta-Sheet Structure. These data show that the supramolecular Structure of amyloid fibrils is not determined by the amino acid sequence at the level of 7-residue or 15-residue segments. The proton-mediated 2D exchange spectra additionally demonstrate that the intermolecular alignment in the beta-Sheets of these amyloid fibrils is highly ordered, with no detectable evidence for "misalignment" defects.
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constraints on supramolecular Structure in amyloid fibrils from two dimensional solid state nmr spectroscopy with uniform isotopic labeling
Journal of the American Chemical Society, 2003Co-Authors: Robert Tycko, Yoshitaka IshiiAbstract:We show that strong constraints on supramolecular Structure in amyloid fibrils can be obtained from solid-state nuclear magnetic resonance measurements on samples with uniformly 13C-labeled segments. The measurements exploit two-dimensional (2D) 13C−13C exchange spectroscopy in conjunction with high-speed magic angle spinning, with proton-mediated exchange of 13C nuclear spin magnetization as recently demonstrated by Baldus and co-workers (J. Am. Chem. Soc. 2002, 124, 9704−9705). Proton-mediated 2D exchange spectra of fibrils formed by residues 16−22 of the 40-residue Alzheimer's β-amyloid peptide show strong nonsequential, intermolecular cross-peaks between α-carbons that dictate an antiparallel β-Sheet Structure in which residue 16+k aligns with residue 22−k. The strong α/α cross-peaks are absent from conventional, direct 2D exchange spectra. Proton-mediated 2D exchange spectra of fibrils formed by residues 11−25 indicate an antiparallel β-Sheet Structure with a pH-dependent intermolecular alignment. In...
