John J. Balbach

A structural model for Alzheimer's β-amyloid fibrils based on experimental constraints from solid state NMR

We present a structural model for amyloid fibrils formed by the 40-residue β-amyloid peptide associated with Alzheimers disease (Aβ1–40), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-β structural motif established by x-ray fiber diffraction and satisfies constraints on Aβ1–40 fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Aβ1–40 are structurally disordered in the fibrils. Residues 12–24 and 30–40 adopt β-strand conformations and form parallel β-sheets through intermolecular hydrogen bonding. Residues 25–29 contain a bend of the peptide backbone that brings the two β-sheets in contact through sidechain-sidechain interactions. A single cross-β unit is then a double-layered β-sheet structure with a hydrophobic core and one hydrophobic face. The only charged sidechains in the core are those of D23 and K28, which form salt bridges. Fibrils with minimum mass-per-length and diameter consist of two cross-β units with their hydrophobic faces juxtaposed.

Supramolecular Structure in Full-Length Alzheimer's β-Amyloid Fibrils: Evidence for a Parallel β-Sheet Organization from Solid-State Nuclear Magnetic Resonance

Abstract We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue β -amyloid peptide associated with Alzheimers disease (A β 1–40 ) obtained from solid-state nuclear magnetic resonance (NMR) measurements of intermolecular dipole-dipole couplings between 13 C labels at 11 carbon sites in residues 2 through 39. The measurements are carried out under magic-angle spinning conditions, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) technique. We also present one-dimensional 13 C magic-angle spinning NMR spectra of the labeled A β 1–40 samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8±0.5A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly β -sheet structure of the amyloid fibrils. The one-dimensional NMR spectra indicate structural order at these sites. The fpRFDR-CT data and NMR spectra also indicate structural disorder in the N-terminal segment of A β 1–40 , including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length β -amyloid fibrils.

Increasing the Amphiphilicity of an Amyloidogenic Peptide Changes the β-Sheet Structure in the Fibrils from Antiparallel to Parallel

Solid-state NMR measurements have been reported for four peptides derived from beta-amyloid peptide Abeta(1-42): Abeta(1-40), Abeta(10-35), Abeta(16-22), and Abeta(34-42). Of these, the first two are predicted to be amphiphilic and were reported to form parallel beta-sheets, whereas the latter two peptides appear nonamphiphilic and adopt an antiparallel beta-sheet organization. These results suggest that amphiphilicity may be significant in determining fibril structure. Here, we demonstrate that acylation of Abeta(16-22) with octanoic acid increases its amphiphilicity and changes the organization of fibrillar beta-sheet from antiparallel to parallel. Electron microscopy, Congo Red binding, and one-dimensional 13C NMR measurements demonstrate that octanoyl-Abeta(16-22) forms typical amyloid fibrils. Based on the stability of monolayers at the air-water interface, octanoyl-Abeta(16-22) is more amphiphilic than Abeta(16-22). Measurements of 13C-13C and 15N-13C nuclear magnetic dipole-dipole couplings in isotopically labeled fibril samples, using the constant-time finite-pulse radiofrequency-driven recoupling (fpRFDR-CT) and rotational echo double resonance (REDOR) solid-state NMR techniques, demonstrate that octanoyl-Abeta(16-22) fibrils are composed of parallel beta-sheets, whereas Abeta(16-22) fibrils are composed of antiparallel beta-sheets. These data demonstrate that amphiphilicity is critical in determining the structural organization of beta-sheets in the amyloid fibril. This work also shows that all amyloid fibrils do not share a common supramolecular structure, and suggests a method for controlling the structure of amyloid fibrils.

Site-Specific Identification of Non-β-Strand Conformations in Alzheimer's β-Amyloid Fibrils by Solid-State NMR

The most well-established structural feature of amyloid fibrils is the cross-beta motif, an extended beta-sheet structure formed by beta-strands oriented perpendicular to the long fibril axis. Direct experimental identification of non-beta-strand conformations in amyloid fibrils has not been reported previously. Here we report the results of solid-state NMR measurements on amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimers disease (Abeta(1-40)), prepared synthetically with pairs of (13)C labels at consecutive backbone carbonyl sites. The measurements probe the peptide backbone conformation in residues 24-30, a segment where a non-beta-strand conformation has been suggested by earlier sequence analysis, cross-linking experiments, and molecular modeling. Data obtained with the fpRFDR-CT, DQCSA, and 2D MAS exchange solid-state NMR techniques, which provide independent constraints on the phi and psi backbone torsion angles between the labeled carbonyl sites, indicate non-beta-strand conformations at G25, S26, and G29. These results represent the first site-specific identification and characterization of non-beta-strand peptide conformations in an amyloid fibril.

Measurement of dipole-coupled lineshapes in a many-spin system by constant-time two-dimensional solid state NMR with high-speed magic-angle spinning

Abstract A two-dimensional solid state NMR technique for measurements of dipole–dipole couplings in many-spin systems under high-speed magic-angle spinning (MAS) is described. The technique, called constant-time finite-pulse radio-frequency-driven recoupling (fpRFDR-CT), uses the fpRFDR pulse sequence to generate non-zero effective homonuclear dipole–dipole couplings under high-speed MAS that have the same operator symmetry as static dipole–dipole couplings. By incorporating fpRFDR into a multiple-pulse cycle based on the Waugh–Huber–Haeberlen (WAHUHA) homonuclear decoupling cycle, a constant-time t 1 evolution period is created. The constant-time t 1 period minimizes distortions of the experimental data due to various pulse sequence imperfections. The fpRFDR-CT technique is demonstrated experimentally in 13 C NMR spectroscopy of carboxylate-labeled, polycrystalline l -alanine. 2D fpRFDR-CT spectra correlate the dipole-coupled lineshape of the 13 C carboxylate groups with their isotropic chemical shift. Good agreement is obtained between the experimental second and fourth moments of the dipole-coupled lineshapes and calculated moments based on the l -alanine crystal structure and an average Hamiltonian analysis of the fpRFDR sequence. Applications in structural investigations of biologically relevant systems are anticipated. This technique illustrates many of the important concepts in modern multi-dimensional solid state NMR.

High-resolution NMR in inhomogeneous fields

Abstract A new NMR technique is presented that yields high-resolution, 1-D NMR spectra of solutes in inhomogeneous magnetic fields. The method exploits the nuclear Overhauser effect which couples the longitudinal relaxation of solvent and solute nuclear spins. Effectively, the solvent spins serve as reporters or local gaussmeters for the solute spins. Both 2-D and 1-D versions of this new experiment are reported. Potential applications of the method include in vivo NMR spectroscopy, where the field homogeneity is alwats degraded by the magnetic susceptibilities of the various tissues.