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Biomedical subjects

Robert Tycko

Publications and source records attributed to Robert Tycko.

33 records · Page 2Linked to original sources
PubMed2004

Broadband rotational resonance in solid state NMR spectroscopy.

A new technique for restoring nuclear magnetic dipole-dipole couplings under magic-angle spinning (MAS) in solid state nuclear magnetic resonance (NMR) spectroscopy is described and demonstrated. In this technique, called broadband rotational resonance (BroBaRR), the coupling between a pair of nuclear spins with NMR frequency difference close (but not necessarily equal) to the MAS frequency is restored by the application of a train of weak radio-frequency pulses at a carrier frequency close to the average of the two NMR frequencies. Phase or amplitude modulation of the pulse train at half the MAS frequency splits the carrier into sidebands close to the two NMR frequencies. The pulse train then removes offsets from the exact rotational resonance condition, leading to dipolar recoupling over a bandwidth controlled by the amplitude of the pulse train. (13)C NMR experiments on uniformly (15)N,(13)C-labeled L-valineHClH(2)O powder validate the theoretical analysis. BroBaRR will be useful in studies of molecular structures by solid state NMR, for example in the detection of long-range couplings between carbons in uniformly labeled organic and biological materials.

Journal Article↗
PubMed2004

Solid-state NMR yields structural constraints on the V3 loop from HIV-1 Gp120 bound to the 447-52D antibody Fv fragment.

Solid-state NMR measurements were performed on the complex of an 18-residue peptide derived from the V3 loop sequence of the gp120 envelope glycoprotein of the HIV-1 MN strain with Fv fragments of the human anti-gp120 monoclonal antibody 447-52D in a frozen glycerol/water solution. The peptide was uniformly (15)N- and (13)C-labeled in a 7-residue segment containing the conserved GPGR motif in the epitope. (15)N and (13)C NMR chemical shift assignments for the labeled segment were obtained from two-dimensional (13)C-(13)C and (15)N-(13)C magic-angle spinning NMR spectra. Reductions in (13)C NMR line widths and changes in chemical shifts upon complex formation indicate the adoption of a well-defined, antibody-dependent structure. Intramolecular (13)C-(13)C distances in the complex, which constrain the peptide backbone and side chain conformations in the GPGR motif, were determined from an analysis of rotational resonance (RR) data. Structural constraints from chemical shifts and RR measurements are in good agreement with recent solution NMR and crystallographic studies of this system, although differences regarding structural ordering of certain peptide side chains are noted. These experiments explore and help delineate the utility of solid state NMR techniques as structural probes of peptide/protein complexes in general, potentially including membrane-associated hormone/receptor complexes.

Amino Acid Sequence↗
PubMed2004

Absolute structural constraints on amyloid fibrils from solid-state NMR spectroscopy of partially oriented samples.

We demonstrate that absolute, molecular-level structural information can be obtained from solid-state NMR measurements on partially oriented amyloid fibrils. Specifically, we show that the direction of the fibril axis relative to a carbonyl 13C chemical shift anisotropy (CSA) tensor can be determined from magic-angle spinning (MAS) sideband patterns in 13C NMR spectra of fibrils deposited on planar substrates. Deposition of fibrils on a planar substrate creates a highly anisotropic distribution of fibril orientations (hence, CSA tensor orientations) with most fibrils lying in the substrate plane. The anisotropic orientational distribution gives rise to distorted spinning sideband patterns in MAS spectra from which the fibril axis direction can be inferred. The experimentally determined fibril axis direction relative to the carbonyl CSA tensor of Val12 in fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta1-40) agrees well with the predictions of a recent structural model (Petkova et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742-16747) in which Val12 is contained in a parallel beta-sheet in the cross-beta motif characteristic of amyloid fibrils.

Amyloid↗
PubMed2004

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment.

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.

Anti-HIV Agents↗
PubMed2004

Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems.

We describe investigations of the effects of rotational resonance (R(2)) on solid state (13)C NMR spectra of uniformly (13)C-labeled samples obtained under magic-angle spinning (MAS), and of the utility of R(2) measurements as structural probes of peptides and proteins with multiple uniformly labeled residues. We report results for uniformly (13)C-labeled L-alanine and L-valine in polycrystalline form, and for amyloid fibrils formed by the 15-residue peptide A beta(11-25) with uniform labeling of a four-residue segment. The MAS NMR spectra reveal a novel J-decoupling effect at R(2) conditions that may be useful in spectral assignments for systems with sharp (13)C MAS NMR lines. Pronounced dependences of the apparent isotropic (13)C NMR chemical shifts on MAS frequency near R(2) conditions are also observed. We demonstrate the feasibility of quantitative (13)C-(13)C distance determinations in L-valine, and qualitative determinations of inter-residue (13)C-(13)C contacts in A beta(11-25) fibrils. Finally, we demonstrate a "relayed" R(2) technique that may be useful in structural measurements on systems with poorly resolved (13)C MAS NMR lines.

Alanine↗
PubMed2004

Progress towards a molecular-level structural understanding of amyloid fibrils.

The problem of determining and understanding the molecular structures of amyloid fibrils has attracted considerable attention and effort over the past several years. Although complete, high-resolution structures have not yet been obtained, key features of protein and peptide conformations and supramolecular organization within amyloid fibrils have been elucidated using a variety of novel experimental methods, including magnetic resonance spectroscopies, electron microscopy, X-ray and neutron scattering, and biochemical techniques. The experimental data are beginning to shed light on issues such as the nature of the intermolecular interactions that stabilize amyloid structures, the molecular structural basis for polymorphism in amyloid fibrils, the universality of amyloid structures, and the balance between structural order and disorder within amyloid fibrils. Recent structural data will contribute to an improved understanding of the mechanisms of amyloid formation and to the development of therapeutic agents for amyloid diseases.

Amyloid↗
PubMed2004

Increasing the amphiphilicity of an amyloidogenic peptide changes the beta-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.

Acylation↗
PubMed2003

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.

Carbon Isotopes↗
PubMed2003

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.

Amyloid↗
PubMed2003

Site-specific identification of non-beta-strand conformations in Alzheimer's beta-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 Alzheimer's 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.

Alzheimer Disease↗
PubMed2002

Supramolecular structural constraints on Alzheimer's beta-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance.

We describe electron microscopy (EM), scanning transmission electron microscopy (STEM), and solid-state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the 42-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1)(-)(42)) and by residues 10-35 of the full-length peptide (Abeta(10)(-)(35)). These measurements place constraints on the supramolecular structure of the amyloid fibrils, especially the type of beta-sheets present in the characteristic amyloid cross-beta structural motif and the assembly of these beta-sheets into a fibril. EM images of negatively stained Abeta(10)(-)(35) fibrils and measurements of fibril mass per length (MPL) by STEM show a strong dependence of fibril morphology and MPL on pH. Abeta(10)(-)(35) fibrils formed at pH 3.7 are single "protofilaments" with MPL equal to twice the value expected for a single cross-beta layer. Abeta(10)(-)(35) fibrils formed at pH 7.4 are apparently pairs of protofilaments or higher order bundles. EM and STEM data for Abeta(1)(-)(42) fibrils indicate that protofilaments with MPL equal to twice the value expected for a single cross-beta layer are also formed by Abeta(1)(-)(42) and that these protofilaments exist singly and in pairs at pH 7.4. Solid-state NMR measurements of intermolecular distances in Abeta(10)(-)(35) fibrils, using multiple-quantum (13)C NMR, (13)C-(13)C dipolar recoupling, and (15)N-(13)C dipolar recoupling techniques, support the in-register parallel beta-sheet organization previously established by Lynn, Meredith, Botto, and co-workers [Benzinger et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 13407-13412; Benzinger et al. (2000) Biochemistry 39, 3491-3499] and show that this beta-sheet organization is present at pH 3.7 as well as pH 7.4 despite the differences in fibril morphology and MPL. Solid-state NMR measurements of intermolecular distances in Abeta(1)(-)(42) fibrils, which represent the first NMR data on Abeta(1)(-)(42) fibrils, also indicate an in-register parallel beta-sheet organization. These results, along with previously reported data on Abeta(1)(-)(40) fibrils, suggest that the supramolecular structures of Abeta(10)(-)(35), Abeta(1)(-)(40), and Abeta(1)(-)(42) fibrils are quite similar. A schematic structural model of these fibrils, consistent with known experimental EM, STEM, and solid-state NMR data, is presented.

Alzheimer Disease↗
PubMed2002

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

We present a structural model for amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta(1-40)), based on a set of experimental constraints from solid state NMR spectroscopy. The model additionally incorporates the cross-beta structural motif established by x-ray fiber diffraction and satisfies constraints on Abeta(1-40) fibril dimensions and mass-per-length determined from electron microscopy. Approximately the first 10 residues of Abeta(1-40) are structurally disordered in the fibrils. Residues 12-24 and 30-40 adopt beta-strand conformations and form parallel beta-sheets through intermolecular hydrogen bonding. Residues 25-29 contain a bend of the peptide backbone that brings the two beta-sheets in contact through sidechain-sidechain interactions. A single cross-beta unit is then a double-layered beta-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-beta units with their hydrophobic faces juxtaposed.

Amino Acid Sequence↗
PubMed2002

Sensitivity enhancement in structural measurements by solid state NMR through pulsed spin locking.

Free induction decay (FID) signals in solid state NMR measurements performed with magic angle spinning can often be extended in time by factors on the order of 10 by a simple pulsed spin locking technique. The sensitivity of a structural measurement in which the structural information is contained in the dependence of the integrated FID amplitude on a preceding evolution period can therefore be enhanced substantially by pulsed spin locking in the signal detection period. We demonstrate sensitivity enhancements in a variety of solid state NMR techniques that are applicable to selectively isotopically labeled samples, including 13C-15N rotational echo double resonance (REDOR), 13C-13C dipolar recoupling measurements using the constant-time finite-pulse radio-frequency-driven recoupling (fpRFDR-CT) and constant-time double-quantum-filtered dipolar recoupling (CTDQFD) techniques, and torsion angle measurements using the double quantum chemical shift anisotropy (DQCSA) technique. Further, we demonstrate that the structural information in the solid state NMR data is not distorted by pulsed spin locking in the detection period.

Amino Acids↗
PubMed2002

Supramolecular structure in full-length Alzheimer's beta-amyloid fibrils: evidence for a parallel beta-sheet organization from solid-state nuclear magnetic resonance.

We report constraints on the supramolecular structure of amyloid fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (A beta(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 beta(1-40) samples. The fpRFDR-CT data reveal nearest-neighbor intermolecular distances of 4.8 +/- 0.5 A for carbon sites from residues 12 through 39, indicating a parallel alignment of neighboring peptide chains in the predominantly beta-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 beta(1-40), including the first nine residues. These results place strong constraints on any molecular-level structural model for full-length beta-amyloid fibrils.

Alzheimer Disease↗