Structural evolution of Iowa mutant β-amyloid fibrils from polymorphic to homogeneous states under repeated seeded growth

Structural variations in β-amyloid fibrils are potentially important to the toxicity of these fibrils in Alzheimer's disease (AD). We describe a repeated seeding protocol that selects a homogeneous fibril structure from a polymorphic initial state in the case of 40-residue β-amyloid fibrils with the Asp23-to-Asn, or Iowa, mutation (D23N-Aβ(1-40)). We use thioflavin T (ThT) fluorescence, transmission electron microscopy (TEM), and solid-state nuclear magnetic resonance (NMR) to track the evolution of fibril structure through multiple generations under this protocol. The data show that (i) repeated seeding selectively amplifies a single D23N-Aβ(1-40) fibril structure that can be a minor component of the initial polymorphic state; (ii) the final structure is highly sensitive to growth conditions, including pH, temperature, and agitation; (iii) although the initial state can include fibrils that contain both antiparallel and parallel β-sheets, the final structures contain only parallel β-sheets, suggesting that antiparallel β-sheet structures are thermodynamically and kinetically metastable. Additionally, our data demonstrate that ThT fluorescence enhancements, which are commonly used to monitor amyloid fibril formation, vary strongly with structural variations, even among fibrils comprised of the same polypeptide. Finally, we present a simple mathematical model that describes the structural evolution of fibril samples under repeated seeding.     

Investigating the structural properties of amyloid-like fibrils formed in vitro from beta2-microglobulin using limited proteolysis and electrospray ionisation mass spectrometry

The protein beta(2)-microglobulin (beta(2)m) aggregates to form classical amyloid fibrils in patients undergoing long-term haemodialysis. Amyloid-like fibrils with a cross-beta fold can also be formed from wild-type beta(2)m under acidic conditions in vitro. The morphology of such fibrils depends critically on the conditions used: incubation of beta(2)m in low ionic strength buffers at pH 2.5 results in the formation of long (microm), straight fibrils while, at pH 3.6, short (<500 nm) fibrils form. At higher ionic strengths (0.2-0.4 M) at pH 1.5-3.6, the fibrils have a distinct curved and nodular morphology. To determine the conformational properties of beta(2)m within in vitro fibrils of different morphologies, limited proteolysis of each fibril type using pepsin was performed and the resulting peptide fragments identified by tandem mass spectrometry. For comparison, the proteolytic degradation patterns of monomeric beta(2)m and seven synthetic peptides spanning the entire sequence of the intact protein were similarly analysed. The results show that fibrils with different morphologies result in distinct digestion patterns. While the curved, worm-like fibrils are relatively weakly protected from proteolysis, the long, straight fibrils formed at pH 2.5 at low ionic strength show only a single cut-site at Val9, demonstrating that substantial refolding of the initially acid-denatured and unprotected state of beta(2)m occurs during assembly. The data demonstrate that the organisation of the polypeptide chain in fibrils with different morphological features differs considerably, despite the fact that the fibrils possess a common cross-beta architecture.     

Constraints on supramolecular structure in amyloid fibrils from two-dimensional solid-state NMR spectroscopy with uniform isotopic labeling

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.     

Intermolecular alignment in Y145Stop human prion protein amyloid fibrils probed by solid-state NMR spectroscopy

The Y145Stop mutant of human prion protein, huPrP23-144, has been linked to PrP cerebral amyloid angiopathy, an inherited amyloid disease, and also serves as a valuable in vitro model for investigating the molecular basis of amyloid strains. Prior studies of huPrP23-144 amyloid by magic-angle-spinning (MAS) solid-state NMR spectroscopy revealed a compact β-rich amyloid core region near the C-terminus and an unstructured N-terminal domain. Here, with the focus on understanding the higher-order architecture of huPrP23-144 fibrils, we probed the intermolecular alignment of β-strands within the amyloid core using MAS NMR techniques and fibrils formed from equimolar mixtures of (15)N-labeled protein and (13)C-huPrP23-144 prepared with [1,3-(13)C(2)] or [2-(13)C]glycerol. Numerous intermolecular correlations involving backbone atoms observed in 2D (15)N-(13)C spectra unequivocally suggest an overall parallel in-register alignment of the β-sheet core. Additional experiments that report on intermolecular (15)N-(13)CO and (15)N-(13)Cα dipolar couplings yielded an estimated strand spacing that is within ～10% of the distances of 4.7-4.8 ? typical for parallel β-sheets.     

Molecular-level examination of Cu2+ binding structure for amyloid fibrils of 40-residue Alzheimer's β by solid-state NMR spectroscopy

Cu(2+) binding to Alzheimer's β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.     

Local cooperativity in an amyloidogenic state of human lysozyme observed at atomic resolution

The partial unfolding of human lysozyme underlies its conversion from the soluble state into amyloid fibrils observed in a fatal hereditary form of systemic amyloidosis. To understand the molecular origins of the disease, it is critical to characterize the structural and physicochemical properties of the amyloidogenic states of the protein. Here we provide a high-resolution view of the unfolding process at low pH for three different lysozyme variants, the wild-type protein and the mutants I56T and I59T, which show variable stabilities and propensities to aggregate in vitro. Using a range of biophysical techniques that includes differential scanning calorimetry and nuclear magnetic resonance spectroscopy, we demonstrate that thermal unfolding under amyloidogenic solution conditions involves a cooperative loss of native tertiary structure, followed by progressive unfolding of a compact, molten globule-like denatured state ensemble as the temperature is increased. The width of the temperature window over which the denatured ensemble progressively unfolds correlates with the relative amyloidogenicity and stability of these variants, and the region of lysozyme that unfolds first maps to that which forms the core of the amyloid fibrils formed under similar conditions. Together, these results present a coherent picture at atomic resolution of the initial events underlying amyloid formation by a globular protein.     

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.     

Solid-state NMR spectroscopy method for determination of the backbone torsion angle psi in peptides with isolated uniformly labeled residues

We demonstrate a solid-state nuclear magnetic resonance technique, with the acronym ROCSA-LG, for the determination of backbone torsion angles psi in peptides with multiple, but isolated, uniformly labeled residues. The method correlates the 13C' chemical shift anisotropy and the 13Calpha-1Halpha heteronuclear dipolar tensors within a single uniformly labeled residue in a two-dimensional (2D) experiment. The technique requires the measurement of only five 2D spectra and is compatible with high-speed magic-angle spinning. Experimental results are presented for the 17-residue alpha-helical peptide MB(i+4)EK and for amyloid fibrils formed by the 15-residue peptide Abeta11-25.     

A designed protein interface that blocks fibril formation

Protein fibril formation is implicated in many diseases, and therefore much effort has been focused toward the development of inhibitors of this process. In a previous project, a monomeric protein was computationally engineered to bind itself and form a heterodimer complex following interfacial redesign. One of the protein monomers, termed monomer-B, was unintentionally destabilized and shown to form macroscopic fibrils. Interestingly, in the presence of the designed binding partner, fibril formation was blocked. Here we describe the complete characterization of the amyloid properties of monomer-B and the inhibition of fiber formation by the designed binding partner, monomer-A. Both proteins are mutants of the betal domain of streptococcal protein-G. The free monomer-B protein forms amyloid-type fibrils, as determined by transmission electron microscopy and the change in fluorescence of Thioflavin T, an amyloid-specific dye. Fibril formation kinetics are influenced by pH, protein concentration, and seeding with preformed fibrils. Under all conditions tested, monomer-A was able to inhibit the formation of monomer-B fibrils. This inhibition is specific to the engineered interaction, as incubation of monomer-B with wild-type protein-G (a structural homologue) did not result in inhibition under the same conditions. Thus, this de novo-designed heterodimeric complex is an excellent model system for the study of protein-based fibril formation and inhibition. This system provides additional insight into the development of pharmaceuticals for amyloid disorders, as well as the potential use of amyloid fibrils for self-assembling nanostructures.     

Interaction between synthetic amyloid-beta-peptide (1-40) and its aggregation inhibitors studied by electrospray ionization mass spectrometry.

It is generally postulated that amyloid-beta-peptides play a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathological properties of these peptides, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of amyloid-beta-peptides to form beta-sheet structures and/or amyloid fibrils. Amyloid-beta-peptides are known to aggregate spontaneously in vitro with the formation of amyloid fibrils. The intervention on the amyloid-beta-peptides aggregation process can be envisaged as an approach to stopping or slowing the progression of Alzheimer's disease. In the last few years a number of small molecules have been reported to interfere with the in vitro aggregation of amyloid-beta-peptides. Melatonin, a hormone recently found to protect neurons against amyloid-beta-peptide toxicity, interacts with amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and inhibits the progressive formation of beta-sheet and/or amyloid fibrils. These interactions between melatonin and the amyloid peptides have been demonstrated by circular dichroism (CD) and electron microscopy for amyloid-beta-peptide (1-40) and amyloid-beta-peptide (1-42) and by nuclear magnetic resonance (NMR) spectroscopy for amyloid-beta-peptide (1-40). Our electrospray ionization mass spectrometric (ESI-MS) studies also proved that there is a hydrophobic interaction between amyloid-beta-peptide (1-40) and melatonin and the proteolytic investigations suggested that the interaction took place on the 29-40 amyloid-beta-peptide segment. The wide-ranging application of these results would provide further information and help in biological research.     

Theoretical investigations of TTR derived aggregation-prone peptides’ potential to biochemically attenuate the amyloidogenic propensities of V30 M TTR amyloid fibrils

Transthyretin (TTR) is a cerebrospinal fluid and plasma prevalent protein implicated in heritable and sporadic amyloidosis. Numerous mutations and a wide range of phenotypes have been associated with TTR-mediated amyloidosis. Among these, V30 M is the most predominant point mutation, inculpated with familial amyloid polyneuropathy (FAP), a life-threatening autosomal dominant genetic disorder characterized by the deposition of amyloid fibrils in crucial areas. Hence, efficacious therapeutics against this detrimental disorder is warranted. Lately, several peptide-based analeptics, especially the ones that are aggregation-prone and the ones derived from aggregation hotspots of amyloidogenic proteins are being increasingly proffered against the amyloid fibrils. In the present study, as an effective precursor to in vitro investigations, we examined and assessed the therapeutic potentials of aggregation-prone peptides (APPs) derived from TTR, against V30 M TTR amyloid fibrils, computationally. Out of five experimentally corroborated APPs availed for this study, molecular dynamics simulation analysis endorses APP TAVVTN to be an effective beta-sheet breaker against V30 M TTR amyloid fibrils. Furthermore, consistent findings from various molecular trajectory analyses, residual frustration analysis and simulated thermal denaturation have indicated that APP TAVVTN could effectually relater the structural dynamics of V30 M TTR amyloid fibrils, to conformationally digress it away from its amyloidogenic propensities. Hence, based on consistent unvarying findings from numerous adept computational pipelines, APP TAVVTN could be an efficacious analeptic to therapeutically intervene and mitigate the amyloidogenic propensities of V30 M TTR amyloid fibrils, thereby ameliorating the pathological ramifications due to FAP.     

beta Sheet structure in amyloid beta fibrils and vibrational dipolar coupling

Fibrils formed by amyloid beta proteins were labeled with 13C at various positions and examined by infrared spectroscopy to detect vibrational dipolar coupling, implying close physical proximity. The results support key features of several recently proposed models for amyloid fibril structure, but they also add some important caveats. For instance, they support the conclusion that the beta structure is parallel; however, the coupling is not as strong as expected when residues are in register. This may be explained by out-of-register alignment of adjacent strands, or nonstandard parallel sheet structure that yields suboptimal alignment of labeled dipole moments. The data also point to a significant structural difference between fibrils formed by the 40-residue amyloid beta protein and fibrils formed by residues 10-35.     

Microtubule‐Binding R3 Fragment from Tau Self‐Assembles into Giant Multistranded Amyloid Ribbons

Tau protein and its fragments self‐assemble into amyloid fibrils in the presence of polyanions, such as heparin. By combining microscopy, scattering, and spectroscopy techniques, we studied the aggregation of the 26‐mer Tau‐derived peptide alone, Tau_306–327, the third repeat fragment (R3) of the microtubule‐binding domain. We show that: i) the sole Tau_306–327 can self‐assemble into amyloid fibrils without the need of aggregation‐promoting polyanions; ii) the resulting structures consist of surprisingly large, well‐ordered 2D laminated flat ribbons, with a log‐normal distribution of the lateral width, reaching the unprecedented lateral size of 350 nm and/or 45 individual protofilaments, that is, the largest amyloid laminated structures ever observed for Tau or any other amyloidogenic sequence. Our results provide insight into the molecular determinants of Tau aggregation and open new perspectives in the understanding of the assembly of amyloid fibrils and β‐sheet‐based biomaterials.     

Environmental factors differently affect human and rat IAPP: conformational preferences and membrane interactions of IAPP17-29 peptide derivatives

Interest in the 37-residue human islet amyloid polypeptide (hIAPP) is related to its ability to form amyloid deposits in patients affected by type II diabetes. Attempts to unravel the molecular features of this disease have indicated several regions of this polypeptide to be responsible for either the ability to form insoluble fibrils or the abnormal interaction with membranes. To extend these studies to peptides that enclose His18, whose ionization state is believed to play a key role in the aggregation of hIAPP, we report on the synthesis of two peptides, hIAPP17-29 and rIAPP17-29, encompassing the 17-29 sequences of human and rat IAPP, respectively, as well as on their conformational features in water and in several membrane-mimicking environments as revealed by circular dichroism (CD) and 2D-NMR studies. hIAPP17-29 adopts a beta-sheet structure in water and its solubility increases at low pH. Anionic sodium dodecyl sulfate (SDS) micelles promoted the formation of an alpha-helical structure in the peptide chain, which was poorly influenced by pH variations. rIAPP17-29 was soluble and unstructured in all the environments investigated, with a negligible effect of pH. The membrane interactions of hIAPP17-29 and rIAPP17-29 were assessed by recording differential scanning calorimetry (DSC) measurements aimed at elucidating the peptide-induced changes in the thermotropic behaviour of zwitterionic (DPPC) and negatively charged (DPPC/DPPS 3:1) model membranes (DPPC=1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPS=1,2-dipalmitoyl-sn-glycero-3-phosphoserine). Results of DSC experiments demonstrated the high potential of hIAPP17-29 to interact with DPPC membranes. hIAPP17-29 exhibited a negligible affinity for negatively charged DPPC/DPPS model membranes at neutral pH. On the other hand, rIAPP17-29 did not interact with neutral or negatively charged membranes. The role played by His18 in the modulation of the biophysical properties of this hIAPP region was assessed by synthesising and studying the R18HrIAPP17-29 peptide; the replacement of a single Arg with a His residue is not sufficient to induce either amyloidogenic propensity or membrane interaction in this region. The results show that the 17-29 domain of hIAPP has many properties of the full-length protein "in vitro" and this opens up new perspectives for both research and eventually therapy.     

Inactivation of nitroalkane oxidase upon mutation of the active site base and rescue with a deprotonated substrate

Mutation of Asp402 in nitroalkane oxidase to Asn or Ala inactivates the enzyme with neutral nitroethane as substrate, but the activity can be rescued with the nitroethane anion. The V/K values of the D402N and D402A enzymes with the nitroethane anion are independent of pH, whereas the V/K values of the wild-type and D402E enzymes are pH dependent with both the protonated and the deprotonated forms of nitroethane. Moreover, although the V/K value of the D402E enzyme with neutral nitroethane is 20-fold less than that of the wild-type enzyme, there is only a 2-fold difference in the V/K values with the nitroethane anion. These results are fully consistent with a primary role for Asp402 as the active site base in nitroalkane oxidase which abstracts the substrate alpha-proton.     

Changes in interfacial properties of alpha-synuclein preceding its aggregation

Parkinson's disease (PD) is associated with the formation and deposition of amyloid fibrils of the protein alpha-synuclein (AS). It has been proposed that oligomeric intermediates on the pathway to fibrilization rather than the fibrils themselves are the pathogenic agents of PD, but efficient methods for their detection are lacking. We have studied the interfacial properties of wild-type AS and the course of its aggregation in vitro using electrochemical analysis and dynamic light scattering. The oxidation signals of tyrosine residues of AS at carbon electrodes and the ability of fibrils to adsorb and catalyze hydrogen evolution at hanging mercury drop electrodes (HMDEs) decreased during incubation. HMDEs were particularly sensitive to pre-aggregation changes in AS. Already after 1 h of a standard aggregation assay in vitro (stirring at 37 degrees C), the electrocatalytic peak H increased greatly and shifted to less negative potentials. Between 3 and 9 h of incubation, an interval during which dynamic light scattering indicated AS oligomerization, peak H diminished and shifted to more negative potentials, and AS adsorbability decreased. We tentatively attribute the very early changes in the interfacial behavior of the protein after the first few hours of incubation to protein destabilization with disruption of long-range interactions. The subsequent changes can be related to the onset of oligomerization. Our results demonstrate the utility of electrochemical methods as new and simple tools for the investigation of amyloid formation.     

Imaging secondary structure of individual amyloid fibrils of a β2-microglobulin fragment using near-field infrared spectroscopy

Amyloid fibril diseases are characterized by the abnormal production of aggregated proteins and are associated with many types of neuro- and physically degenerative diseases. X-ray diffraction techniques, solid-state magic-angle spinning NMR spectroscopy, circular dichroism (CD) spectroscopy, and transmission electron microscopy studies have been utilized to detect and examine the chemical, electronic, material, and structural properties of amyloid fibrils at up to angstrom spatial resolution. However, X-ray diffraction studies require crystals of the fibril to be analyzed, while other techniques can only probe the bulk solution or solid samples. In the work reported here, apertureless near-field scanning infrared microscopy (ANSIM) was used to probe the secondary structure of individual amyloid fibrils made from an in vitro solution. Simultaneous topographic and infrared images of individual amyloid fibrils synthesized from the #21-31 peptide fragment of β(2)-microglobulin were acquired. Using this technique, IR spectra of the amyloid fibrils were obtained with a spatial resolution of less than 30 nm. It is observed that the experimental scattered field spectrum correlates strongly with that calculated using the far-field absorption spectrum. The near-field images of the amyloid fibrils exhibit much lower scattering of the IR radiation at approximately 1630 cm(-1). In addition, the near-field images also indicate that composition and/or structural variations among individual amyloid fibrils were present.     

Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.     

Molecular Structure of Amyloid Fibrils Formed by Residues 127 to 147 of the Human Prion Protein

Amyloid fibrils are filamentous and insoluble forms of peptides or proteins. Proline has long been considered to be incompatible with the cross‐β structural motif of amyloid fibrils. On the basis of solid‐state NMR spectroscopy data, we present a structural model of an in‐register parallel β sheet for the amyloid fibrils formed from a human prion protein fragment, huPrP_127–47. We have developed a simple solid‐state NMR spectroscopy technique to identify solvent‐protected backbone amide protons in a H/D exchange experiment without disaggregating the amyloid fibrils, from which we find that proline residue P₁₃₇ does not disrupt the β‐sheet structure from G₁₂₇ to G₁₄₂. We suggest that the resultant kink at P₁₃₇ generates a twist between adjacent peptide strands to maintain hydrogen bonding in the β‐sheet regions flanking the P₁₃₇ residue. Although proline can be well integrated into the cross‐β structure of amyloid fibrils, the kink formed at the position of the proline residue will considerably weaken the hydrogen bonding between the neighboring strands, especially when the mutation site is near the central region of a β sheet.     

Structural insights into the polymorphism of amyloid-like fibrils formed by region 20-29 of amylin revealed by solid-state NMR and X-ray fiber diffraction

Many unrelated proteins and peptides can assemble into amyloid or amyloid-like nanostructures, all of which share the cross-beta motif of repeat arrays of beta-strands hydrogen-bonded along the fibril axis. Yet, paradoxically, structurally polymorphic fibrils may derive from the same initial polypeptide sequence. Here, solid-state nuclear magnetic resonance (SSNMR) analysis of amyloid-like fibrils of the peptide hIAPP 20-29, corresponding to the region S (20)NNFGAILSS (29) of the human islet amyloid polypeptide amylin, reveals that the peptide assembles into two amyloid-like forms, (1) and (2), which have distinct structures at the molecular level. Rotational resonance SSNMR measurements of (13)C dipolar couplings between backbone F23 and I26 of hIAPP 20-29 fibrils are consistent with form (1) having parallel beta-strands and form (2) having antiparallel strands within the beta-sheet layers of the protofilament units. Seeding hIAPP 20-29 with structurally homogeneous fibrils from a 30-residue amylin fragment (hIAPP 8-37) produces morphologically homogeneous fibrils with similar NMR properties to form (1). A model for the architecture of the seeded fibrils is presented, based on the analysis of X-ray fiber diffraction data, combined with an extensive range of SSNMR constraints including chemical shifts, torsional angles, and interatomic distances. The model features a cross-beta spine comprising two beta-sheets with an interface defined by residues F23, A25, and L27, which form a hydrophobic zipper. We suggest that the energies of formation for fibril form containing antiparallel and parallel beta-strands are similar when both configurations can be stabilized by a core of hydrophobic contacts, which has implications for the relationship between amino acid sequence and amyloid polymorphism in general.