Solid state NMR reveals a pH-dependent antiparallel beta-sheet registry in fibrils formed by a beta-amyloid peptide.

We report solid state nuclear magnetic resonance (NMR) measurements that probe the supramolecular organization of beta-sheets in the cross-beta motif of amyloid fibrils formed by residues 11-25 of the beta-amyloid peptide associated with Alzheimer's disease (Abeta(11-25)). Fibrils were prepared at pH 7.4 and pH 2.4. The solid state NMR data indicate that the central hydrophobic segment of Abeta(11-25) (sequence LVFFA) adopts a beta-strand conformation and participates in antiparallel beta-sheets at both pH values, but that the registry of intermolecular hydrogen bonds is pH-dependent. Moreover, both registries determined for Abeta(11-25) fibrils are different from the hydrogen bond registry in the antiparallel beta-sheets of Abeta(16-22) fibrils at pH 7.4 determined in earlier solid state NMR studies. In all three cases, the hydrogen bond registry is highly ordered, with no detectable "registry-shift" defects. These results suggest that the supramolecular organization of beta-sheets in amyloid fibrils is determined by a sensitive balance of multiple side-chain-side-chain interactions. Recent structural models for Abeta(11-25) fibrils based on X-ray fiber diffraction data are inconsistent with the solid state NMR data at both pH values.

Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel.

Water-soluble biological macromolecules can be weakly aligned by dissolution in a strained, hydrated gel such as cross-linked polyacrylamide, an effect termed 'strain-induced alignment in a gel' (SAG). SAG induces nonzero nuclear magnetic dipole-dipole couplings that can be measured in high-resolution NMR spectra and used as structural constraints. The dependence of experimental 15N-1H dipolar couplings extracted from two-dimensional heteronuclear single quantum coherence (HSQC) spectra on several properties of compressed polyacrylamide, including the extent of compression, the polyacrylamide concentration, and the cross-link density, is reported for the B1 immunoglobulin binding domain of streptococcal protein G (protein G/B1, 57 residues). It is shown that the magnitude of macromolecular alignment can be widely varied by adjusting these properties, although the orientation and asymmetry of the alignment tensor are not affected significantly. The dependence of the 15N relaxation times T1 and T2 of protein G/B1 on polyacrylamide concentration are also reported. In addition, the results of 15N relaxation and HSQC experiments on the RNA binding domain of prokaryotic protein S4 from Bacillus stearothermophilus (S4 delta41, residues 43-200) in a compressed polyacrylamide gel are presented. These results demonstrate the applicability of SAG to proteins of higher molecular weight and greater complexity. A modified in-phase/anti-phase (IPAP) HSQC technique is described that suppresses natural-abundance 15N background signals from amide groups in polyacrylamide, resulting in cleaner HSQC spectra in SAG experiments. The mechanism of protein alignment in strained polyacrylamide gels is contrasted with that in liquid crystalline media.

Solid-state NMR data support a helix-loop-helix structural model for the N-terminal half of HIV-1 Rev in fibrillar form.

Rev is a 116 residue basic protein encoded by the genome of human immunodeficiency virus type 1 (HIV-1) that binds to multiple sites in the Rev response element (RRE) of viral mRNA transcripts in nuclei of host cells, leading to transport of incompletely spliced and unspliced viral mRNA to the cytoplasm of host cells in the latter phases of the HIV-1 life cycle. Rev is absolutely required for viral replication. Because Rev aggregates and fibrillizes in solution at concentrations required for crystal growth or liquid state NMR measurements, high-resolution structural characterization of full-length Rev has not been possible. Previously, circular dichroism studies have shown that approximately 50 % of the Rev sequence adopts helical secondary structure, predicted to correspond to a helix-loop-helix structural motif in the N-terminal half of the protein. We describe the application of solid-state NMR techniques to Rev fibrils as a means of obtaining site-specific, atomic-level structural constraints without requiring a high degree of solubility or crystallinity. Solid-state NMR measurements, using the double-quantum chemical shift anisotropy and constant-time double-quantum-filtered dipolar recoupling techniques, provide constraints on the phi and psi backbone dihedral angles at sites in which consecutive backbone carbonyl groups are labeled with (13)C. Quantitative analysis of the solid-state NMR data, by comparison with numerical simulations, indicates helical phi and psi angles at residues Leu13 and Val16 in the predicted helix 1 segment, and at residues Arg39, Arg 42, Arg43, and Arg44 in the predicted helix 2 segment. These data represent the first site-specific structural constraints from NMR spectroscopy on full-length Rev, and support the helix-loop-helix structural model for its N-terminal half.

Biomolecular solid state NMR: advances in structural methodology and applications to peptide and protein fibrils.

Solid state nuclear magnetic resonance (NMR) methods can provide atomic-level structural constraints on peptides and proteins in forms that are not amenable to characterization by other high-resolution structural techniques, owing to insolubility, high molecular weight, noncrystallinity, or other characteristics. Important examples include peptide and protein fibrils and membrane-bound peptides and proteins. Recent advances in solid state NMR methodology aimed at structural problems in biological systems are reviewed. The power of these methods is illustrated by experimental results on amyloid fibrils and other protein fibrils.

Multiple quantum solid-state NMR indicates a parallel, not antiparallel, organization of beta-sheets in Alzheimer's beta-amyloid fibrils.

Senile plaques associated with Alzheimer's disease contain deposits of fibrils formed by 39- to 43-residue beta-amyloid peptides with possible neurotoxic effects. X-ray diffraction measurements on oriented fibril bundles have indicated an extended beta-sheet structure for Alzheimer's beta-amyloid fibrils and other amyloid fibrils, but the supramolecular organization of the beta-sheets and other structural details are not well established because of the intrinsically noncrystalline, insoluble nature of amyloid fibrils. Here we report solid-state NMR measurements, using a multiple quantum (MQ) (13)C NMR technique, that probe the beta-sheet organization in fibrils formed by the full-length, 40-residue beta-amyloid peptide (Abeta(1-40)). Although an antiparallel beta-sheet organization often is assumed and is invoked in recent structural models for full-length beta-amyloid fibrils, the MQNMR data indicate an in-register, parallel organization. This work provides site-specific, atomic-level structural constraints on full-length beta-amyloid fibrils and applies MQNMR to a significant problem in structural biology.

Amyloid fibril formation by A beta 16-22, a seven-residue fragment of the Alzheimer's beta-amyloid peptide, and structural characterization by solid state NMR.

The seven-residue peptide N-acetyl-Lys-Leu-Val-Phe-Phe-Ala-Glu-NH(2), called A beta(16-22) and representing residues 16-22 of the full-length beta-amyloid peptide associated with Alzheimer's disease, is shown by electron microscopy to form highly ordered fibrils upon incubation of aqueous solutions. X-ray powder diffraction and optical birefringence measurements confirm that these are amyloid fibrils. The peptide conformation and supramolecular organization in A beta(16-22) fibrils are investigated by solid state (13)C NMR measurements. Two-dimensional magic-angle spinning (2D MAS) exchange and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) measurements indicate a beta-strand conformation of the peptide backbone at the central phenylalanine. One-dimensional and two-dimensional spectra of selectively and uniformly labeled samples exhibit (13)C NMR line widths of <2 ppm, demonstrating that the peptide, including amino acid side chains, has a well-ordered conformation in the fibrils. Two-dimensional (13)C-(13)C chemical shift correlation spectroscopy permits a nearly complete assignment of backbone and side chain (13)C NMR signals and indicates that the beta-strand conformation extends across the entire hydrophobic segment from Leu17 through Ala21. (13)C multiple-quantum (MQ) NMR and (13)C/(15)N rotational echo double-resonance (REDOR) measurements indicate an antiparallel organization of beta-sheets in the A beta(16-22) fibrils. These results suggest that the degree of structural order at the molecular level in amyloid fibrils can approach that in peptide or protein crystals, suggest how the supramolecular organization of beta-sheets in amyloid fibrils can be dependent on the peptide sequence, and illustrate the utility of solid state NMR measurements as probes of the molecular structure of amyloid fibrils. A beta(16-22) is among the shortest fibril-forming fragments of full-length beta-amyloid reported to date, and hence serves as a useful model system for physical studies of amyloid fibril formation.

Solid-state NMR as a probe of amyloid fibril structure.

Amyloid fibrils are intrinsically noncrystalline, insoluble, high-molecular-weight aggregates of peptides and proteins, with considerable biomedical and biophysical significance. Solid-state NMR techniques are uniquely capable of providing high-resolution, site-specific structural constraints for amyloid fibrils, at the level of specific interatomic distances and torsion angles. So far, a relatively small number of solid-state NMR studies of amyloid fibrils have been reported. These have addressed issues about the supramolecular organization of beta-sheets in the fibrils and the peptide conformation in the fibrils, and have concentrated on the beta-amyloid peptide of Alzheimer's disease. Many additional applications of solid-state NMR to amyloid fibrils from a variety of sources are anticipated in the near future, as these systems are ideally suited for the technique and are of widespread current interest.

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements.

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5beta, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with 15N labels at the eta nitrogen positions of arginine side chains and 13C labels at glycine carbonyl positions and 13C-detected 13C-15N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82-85], but is shown by the REDOR measurements to be absent in the RP135/0.5beta complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of phi and psi backbone dihedral angles in the RP135/0.53 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect 13C-15N dipole-dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141-145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331-335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.

Sensitivity enhancement in solid state (15)N NMR by indirect detection with high-speed magic angle spinning.

Enhancement of sensitivity in solid state (15)N NMR by indirect detection through (1)H NMR signals under high-speed magic angle spinning and high-field conditions is demonstrated experimentally on two (15)N-labeled peptides, polycrystalline AlaGlyGly and the helix-forming, 17-residue peptide MB(i + 4)EK in lyophilized form. Sensitivity enhancement factors ranging from 2.0 to 3.2 are observed experimentally, depending on the (15)N and (1)H linewidths and polarization transfer efficiencies. The (1)H-detected two-dimensional (1)H/(15)N correlation spectrum of AlaGlyGly illustrates the possibility of increased spectral resolution and resonance assignments in indirectly detected experiments, in addition to the sensitivity enhancement.

Dual processing of two-dimensional exchange data in magic angle spinning NMR of solids.

We discuss procedures for processing data in rotor-synchronized two-dimensional magic angle spinning (2D MAS) NMR exchange measurements for both structural and dynamical studies. We show, both mathematically and experimentally, that there are two distinct data processing procedures that lead to 2D MAS exchange spectra with purely absorptive crosspeaks. One procedure is that described previously by Hagemeyer, Schmidt-Rohr, and Spiess (HSS). The other procedure is related, but different, and leads to crosspeak intensities given by the formulae of Herzfeld, Roberts, and Griffin (HRG). In 2D MAS exchange experiments on doubly (13)C-labeled l-alanylglycylglycine, we demonstrate that the HSS and HRG crosspeak intensities can be extracted separately from the same data set and contain independent information. Processing and analysis of 2D MAS exchange data with both the HSS and the HRG procedures may enhance utilization of the information content of 2D MAS exchange measurements.

Selection rules for multiple quantum NMR excitation in solids: derivation from time-reversal symmetry and comparison with simulations and (13)C NMR experiments.

New derivations of selection rules for excitation and detection of multiple quantum coherences in coupled spin-1/2 systems are presented. The selection rules apply to experiments in which the effective coupling Hamiltonian used for multiple quantum excitation is both time-reversal invariant and time-reversible by a phase shift of the radiofrequency pulse sequence that generates the effective couplings. The selection rules are shown to be consequences of time-reversal invariance and time-reversibility and otherwise independent of the specific form of the effective coupling Hamiltonian. Numerical simulations of multiple quantum NMR signal amplitudes and experimental multiple quantum excitation spectra are presented for the case of a multiply (13)C-labeled helical polypeptide. The simulations and experiments confirm the selection rules and demonstrate their impact on multiple quantum (13)C NMR spectra in this biochemically relevant case.

Solid-state NMR evidence for an antibody-dependent conformation of the V3 loop of HIV-1 gp120.

Solid-state NMR measurements have been carried out on frozen solutions of the complex of a 24-residue peptide derived from the third variable (V3) loop of the HIV-1 envelope glycoprotein gp120 bound to the Fab fragment of an anti-gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody-bound state. In combination with earlier crystal structures of V3 peptide-antibody complexes and existing data on the cross-reactivity of the antibodies, the solid-state NMR measurements suggest that the Gly-Pro-Gly-Arg (GPGR) motif adopts an antibody-dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full-length gp120. These measurements are the first application of solid-state NMR methods in a structural study of a peptide-protein complex.

Optical pumping in indium phosphide: 31P NMR measurements and potential for signal enhancement in biological solid state NMR.

The initial results of optically-pumped, directly-detected NMR experiments on InP are reported. At low temperatures (4.2 K and above) and in a 9.39 T magnetic field, irradiation of a sample of an undoped InP wafer with 835-nm-wavelength light from a diode laser enhances the spin polarization of 31P nuclei near the sample surface in a manner that depends on the polarization of the light. The nuclear spin polarization is monitored by direct radio-frequency detection of nuclear free induction-decay signals. The maximum nuclear spin polarization (Szn> generated by optical pumping is approximately - 0.004, corresponding to a spin temperature of -0.5 K. The nuclear spin polarization may be limited in these experiments by the use of a high photon energy (1.484 eV) relative to the InP band gap (1.423 eV at low temperatures). It is proposed that optically-pumped InP may be useful as a source of enhanced nuclear spin polarizations for solid state NMR measurements on organic and biological overlayers deposited on InP substrates. Estimates are given for the magnitude of the spin polarization and the efficiency of the polarization transfer from the semiconductor substrate to the overlayer that would be required to permit solid state NMR measurements on sub-nanomole quantities of molecules in the overlayer. These estimates appear well within the range of possibility.

Prospects for resonance assignments in multidimensional solid-state NMR spectra of uniformly labeled proteins.

The feasibility of assigning the backbone 15N and 13C NMR chemical shifts in multidimensional magic angle spinning NMR spectra of uniformly isotopically labeled proteins and peptides in unoriented solid samples is assessed by means of numerical simulations. The goal of these simulations is to examine how the upper limit on the size of a peptide for which unique assignments can be made depends on the spectral resolution, i.e., the NMR line widths. Sets of simulated three-dimensional chemical shift correlation spectra for artificial peptides of varying length are constructed from published liquid-state NMR chemical shift data for ubiquitin, a well-characterized soluble protein. Resonance assignments consistent with these spectra to within the assumed spectral resolution are found by a numerical search algorithm. The dependence of the number of consistent assignments on the assumed spectral resolution and on the length of the peptide is reported. If only three-dimensional chemical shift correlation data for backbone 15N and 13C nuclei are used, no residue-specific chemical shift information, information from amino acid side-chain signals, and proton chemical shift information are available, a spectral resolution of 1 ppm or less is generally required for a unique assignment of backbone chemical shifts for a peptide of 30 amino acid residues.

Electronic states in gallium arsenide quantum wells probed by optically pumped NMR.

An optical pumping technique was used to enhance and localize nuclear magnetic resonance (NMR) signals from an n-doped GaAs/Al0.1Ga0.9As multiple quantum well structure, permitting direct radio-frequency measurements of gallium-71 NMR spectra and nuclear spin-lattice relaxation rates (1/T1) as functions of temperature (1.6 K < T < 4.2 K) and the Landau level filling factor (0.66 < v < 1.76). The measurements reveal effects of electron-electron interactions on the energy levels and spin states of the two-dimensional electron system confined in the GaAs wells. Minima in 1/T1 at v approximately 1 and v approximately 2/3 indicate energy gaps for electronic excitations in both integer and fractional quantum Hall states. Rapid, temperature-independent relaxation at intermediate v values indicates a manifold of low-lying electronic states with mixed spin polarizations.

Molecular dynamics, phase diagrams and electronic properties of fullerenes and alkali fullerides: insights from solid-state nuclear magnetic resonance spectroscopy.

Solid-state nuclear magnetic resonance (NMR) measurements have made important contributions to the current understanding of the structural, dynamical, and electronic properties of fullerenes (C60 and C70) and the alkali fullerides AxC60 (A = alkali metal). These measurements and their interpretation are reviewed. One- and two-dimensional 13C NMR lineshapes and spin-lattice relaxation rates provide evidence for rapid molecular rotations and orientational order-disorder transitions in the fullerenes and alkali fullerides. The kinetics of molecular reorientations are determined from the NMR data. 13C and alkali metal NMR spectra indicate that the alkali fullerides are stoichiometric compounds. Each stable, stoichiometric phase has distinctive NMR signatures. 13C and alkali metal NMR spectra and relaxation measurements provide valuable and unique information about the electronic properties of the metallic, superconducting, and non-metallic phases of the alkali fullerides.

New phases of c60 synthesized at high pressure.

The fullerene C(60) can be converted into two different structures by high pressure and temperature. They are metastable and revert to pristine C(60) on reheating to 300 degrees C at ambient pressure. For synthesis temperatures between 300 degrees and 400 degrees C and pressures of 5 gigapascals, a nominal face-centered-cubic structure is produced with a lattice parameter a(o) = 13.6 angstroms. When treated at 500 degrees to 800 degrees C at the same pressure, C(60) transforms into a rhombohedral structure with hexagonal lattice parameters of a(o) = 9.22 angstroms and c(o) = 24.6 angstroms. The intermolecular distance is small enough that a chemical bond can form, in accord with the reduced solubility of the pressure-induced phases. Infrared, Raman, and nuclear magnetic resonance studies show a drastic reduction of icosahedral symmetry, as might occur if the C(60) molecules are linked.