The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale-including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy-we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils. It is well established that a wide variety of peptides or proteins without any evident sequence similarity can self-assemble into amyloid fibrils (1, 2). These structures have many common characteristics, typically being 100-200 Å in diameter and containing a universal "cross-β" core structure composed of arrays of β-sheets running parallel to the long axis of the fibrils (3). These fibrillar states are highly ordered, with persistence lengths of the order of microns (4) and mechanical properties comparable to those of steel and dragline silk, and much greater than those typical of biological filaments such as actin and microtubules (5). Amyloid fibrils can also possess very high kinetic and thermodynamic stabilities, often exceeding those of the functional folded states of proteins (6), as well as a greater resistance to degradation by chemical or biological means (7). Several functional forms of proteins that exploit these properties have been observed in biological systems (8). More generally, however, the conversion of normally soluble functional proteins into the amyloid state is associated with many debilitating human disorders, ranging from Alzheimer's disease to type II diabetes (1, 9). Our understanding of the nature of this type of filamentous aggregate has greatly improved in recent years (3,(10)(11)(12)(13)(14)(15)(16)(17)(18)(19), particularly through the structural determination of their elementary β-strand building blocks (20) and the characterization of their assembly into cross-β steric zippers (21,22). However, a thorough understanding of the hierarchical assembly of these individual structural elements into fully-formed fibrils, which display polymorphism but possess a range of generic features (23), has so far been limited by the absence of a complete atomicresolution cross-β amyloid structures (2).We report here the simultaneous determination of the a...

We describe three-dimensional magic-angle-spinning NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids. The approaches employ transferred echo double resonance (TEDOR) for (13)C-(15)N coherence transfer and (15)N and (13)C frequency labeling for site-specific resolution, and build on several previous 3D TEDOR techniques. The novel feature of the 3D TEDOR pulse sequences presented here is that they are specifically designed to circumvent the detrimental effects of homonuclear (13)C-(13)C J-couplings on the measurement of weak (13)C-(15)N dipolar couplings. In particular, homonuclear J-couplings lead to two undesirable effects: (i) they generate anti-phase and multiple-quantum (MQ) spin coherences, which lead to spurious cross-peaks and phase-twisted lines in the 2D (15)N-(13)C correlation spectra, and thus degrade the spectral resolution and prohibit the extraction of reliable cross-peak intensities, and (ii) they significantly reduce cross-peak intensities for strongly J-coupled (13)C sites (e.g., CO and C(alpha)). The first experiment employs z-filter periods to suppress the anti-phase and MQ coherences and generates 2D spectra with purely absorptive peaks for all TEDOR mixing times. The second approach uses band-selective (13)C pulses to refocus J-couplings between (13)C spins within the selective pulse bandwidth and (13)C spins outside the bandwidth. The internuclear distances are extracted by using a simple analytical model, which accounts explicitly for multiple spin-spin couplings contributing to cross-peak buildup. The experiments are demonstrated in two U-(13)C,(15)N-labeled peptides, N-acetyl-L-Val-L-Leu (N-ac-VL) and N-formyl-L-Met-L-Leu-L-Phe (N-f-MLF), where 20 and 26 (13)C-(15)N distances up to approximately 5-6 A were measured, respectively. Of the measured distances, 10 in N-ac-VL and 13 in N-f-MLF are greater than 3 A and provide valuable structural constraints.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105-115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13 C and 15 N resonance assignments for TTR(105-115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13 C-15 N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13 C, 15 N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105-115) adopts an extended ␤-strand conformation in the amyloid fibrils such that both the main-and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

We describe a new fivefold symmetric approach to homonuclear recoupling in rotating solids that is based on rotor-synchronized, spin-lock rf irradiation of the type employed previously in MELODRAMA and C7 ͑and their derivative sequences͒ for 13 C-13 C recoupling. The fivefold sequence, like its sevenfold relatives, is ␥-encoded, and therefore exhibits a theoretical efficiency of ϳ73% for double quantum filtering ͑2QF͒. However, since the ratio of rf field strength, rf /2, to spinning frequency, r /2, is lower, it is possible to operate the sequence at higher spinning rates, and we have investigated the 2QF efficiency as a function of 1 H decoupling field strength at high spinning frequencies. We observe dramatic oscillations of the recoupled signal with a period ϳ r /2 indicating that the 1 H reservoir is behaving partially inhomogeneously. This kind of double quantum recoupling is explored in multiple spin systems and we derive analytical forms for polarization transfer and double quantum excitation relevant for uniformly labeled systems. Finally, the wide applicability of the fivefold sequence is demonstrated with INADEQUATE type spectra of uniformly 13 C labeled sucrose and L-alanine.