Jörg Leppert

Structural Basis of β‐Amyloid‐Dependent Synaptic Dysfunctions

Aggregation of b-amyloid (Ab) peptide into oligomers and protofibrils is a hallmark of Alzheimer s disease (AD). Increasing evidence shows that the primary insult in AD is caused by oligomeric species that impair the ordered function of synaptic networks. Consistent with this view, oligomers were shown to affect synaptic plasticity, and they impair the long-term potentiation (LTP) in living brain tissues, a widely used model system of brain memory functions. Using solidstate NMR spectroscopy, we here determined the residuespecific molecular conformation of a highly synaptotoxic bamyloid oligomer structure. Our measurements reveal a stable N-terminal b strand that controls the partitioning between oligomer and protofibril formation, whereas targeting the peptide N-terminus ameliorates Ab-dependent neuronal dysfunctions. The presently investigated, chemically well-defined Ab oligomers faithfully reproduce the hallmark characteristics of AD-related oligomers. Living hippocampal brain slices were exposed to different Ab conformers (Figure 1A), and a series of tetanic electrical stimuli were applied to evoke a longlasting increase of the synaptic transmission, termed LTP. Oligomers, but not freshly dissolved, that is, primarily monomeric, Ab peptide or fibrils, reduce the LTP response and therefore disturb the brain memory functions within these tissue samples (Figure 1B). A similar oligomer-specificity is seen with cultured primary neurons, which present a significant oligomer-dependent decrease ( 40%) of their

Solid state NMR of proteins at high MAS frequencies: symmetry-based mixing and simultaneous acquisition of chemical shift correlation spectra

We have carried out chemical shift correlation experiments with symmetry-based mixing sequences at high MAS frequencies and examined different strategies to simultaneously acquire 3D correlation spectra that are commonly required in the structural studies of proteins. The potential of numerically optimised symmetry-based mixing sequences and the simultaneous recording of chemical shift correlation spectra such as: 3D NCAC and 3D NHH with dual receivers, 3D NC′C and 3D C′NCA with sequential 13C acquisitions, 3D NHH and 3D NC′H with sequential 1H acquisitions and 3D CANH and 3D C’NH with broadband 13C–15N mixing are demonstrated using microcrystalline samples of the β1 immunoglobulin binding domain of protein G (GB1) and the chicken α-spectrin SH3 domain.

MAS solid state NMR of RNAs with multiple receivers.

In the study of biomolecular systems, magic angle spinning (MAS) solid state NMR is emerging as a powerful complementary tool to X-ray crystallography and solution state NMR. Making use of the distance and torsion angle constraints extracted in the solid state the structural characteristics of many biologically interesting systems have been elucidated via MAS solid state NMR recently (Castellani et al. 2002; Jaroniec et al. 2002; Rienstra et al. 2002; Luca et al. 2003; Tycko 2003; Krabben et al. 2004; Zech et al. 2005; Iwata et al. 2006; Egawa et al. 2007; Goldbourt et al. 2007). Homoand heteronuclear distances involving C and N nuclei with well resolved isotropic chemical shifts are commonly used in MAS solid state NMR based structural studies. Although H resonances are generally broad due to strong homonuclear dipolar couplings, the possibilities for extracting short range H–H distance estimates from fully protonated (C, N) labelled peptide/protein samples have been demonstrated recently (de Boer et al. 2002; Lange et al. 2002, 2003, 2005; Tycko and Ishii 2003; Reif et al. 2003). This approach exploits the improved spectral resolution seen in N and C spectra and involves H–H dipolar coupling mediated chemical shift correlation of the low c nuclei. Cross-peak intensities seen in such data, commonly referred to as CHHC, CHHN, NHHN and NHHC spectra, are related to the spatial proximity of the protons that are directly attached to the corresponding nuclei observed in the two dimensions. Our recent studies (Riedel et al. 2005a, 2006) indicate that NHHN, CHHC and NHHC type experiments also hold considerable potential in the structural studies of RNAs that play a critical role in many biological processes and exhibit a variety of secondary and tertiary structural features. Duplex regions arising from consecutive formation of hydrogen bonded base pairs are commonly found in RNA. Hence, the identification of the hydrogen bonded base-pairs and the characterisation of the underlying hydrogen bonding patterns is of critical importance in the study of RNA. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of strong proton–proton dipolar couplings, it has been demonstrated that the characterisation of the hydrogen bonding networks in RNAs can be effectively carried out via NHHN and NHHC type of experiments (Riedel et al. 2005a). In addition, it has also been shown that H–H dipolar coupling mediated C–C chemical shift correlation experiments can facilitate the characterisation of the glycosidic torsion angle v, the sugar pucker and the helical regions of RNAs (Riedel et al. 2006). Hence, data from NHHN, CHHC and NHHC type experiments are critically important in RNA structural studies. Currently, the different proton–proton dipolar coupling mediated N/C chemical shift correlation experiments are carried out individually and, hence, considerable amount of spectrometer time is required to generate data with good signalto-noise ratio. In this communication an efficient approach for the simultaneous collection of these different MAS solid state NMR data sets is presented. The efficacy of the approach is demonstrated using an RNA composed of 97 CUG repeats, (CUG)97, a system that is under investigation Electronic supplementary material The online version of this article (doi:10.1007/s10858-008-9247-1) contains supplementary material, which is available to authorized users.

Structure and biomedical applications of amyloid oligomer nanoparticles.

Amyloid oligomers are nonfibrillar polypeptide aggregates linked to diseases, such as Alzheimers and Parkinsons. Here we show that these aggregates possess a compact, quasi-crystalline architecture that presents significant nanoscale regularity. The amyloid oligomers are dynamic assemblies and are able to release their individual subunits. The small oligomeric size and spheroid shape confer diffusible characteristics, electrophoretic mobility, and the ability to enter hydrated gel matrices or cells. We finally showed that the amyloid oligomers can be labeled with both fluorescence agents and iron oxide nanoparticles and can target macrophage cells. Oligomer amyloids may provide a new biological nanomaterial for improved targeting, drug release, and medical imaging.

Solvent Removal Induces a Reversible β-to-α Switch in Oligomeric Aβ Peptide

Solvation and hydration are key factors for determining the stability and folding of proteins, as well as the formation of amyloid fibrils and related polypeptide aggregates. Using attenuated total reflectance Fourier-transform infrared and solid-state NMR spectroscopy, we find that the Aβ peptide experiences a remarkable conformational switch from β to α secondary structure upon solvent removal by lyophilization of oligomers. This transition is, contrary to Aβ fibrils, independent of concentration of organic co-solvents or co-solutes and is reversible upon re-addition of the solvent. Our data illuminate a previously unnoted secondary structural plasticity of the Aβ peptide in amyloid oligomers that could bear relevance for Aβs interactions with cellular structures of low polarity.

Chemical shift correlation at high MAS frequencies employing low-power symmetry-based mixing schemes

An approach for conveniently implementing low-power CNnν and RNnν symmetry-based band-selective mixing sequences for generating homo- and heteronuclear chemical shift correlation NMR spectra of low γ nuclei in biological solids is demonstrated. Efficient magnetisation transfer characteristics are achieved by selecting appropriate symmetries requiring the application of basic RF elements of relatively long duration and numerically tailoring the RF field modulation profile of the basic element. The efficacy of the approach is experimentally shown by the acquisition of 15N–13C dipolar and 13C–13C scalar and dipolar coupling mediated chemical shift correlation spectra at representative MAS frequencies.

RFDR with Adiabatic Inversion Pulses: Application to Internuclear Distance Measurements

In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.

Recoupling and decoupling of nuclear spin interactions at high MAS frequencies: numerical design of CNnν symmetry-based RF pulse schemes

The CNnν class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic “C” element corresponding to an RF cycle with unity propagator. In this study, the design of CNnν symmetry-based RF pulse sequences for achieving 13C–13C double-quantum dipolar recoupling and through bond scalar coupling mediated 13C–13C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic “C” elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the “C” element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here.

Broadband 15N–13C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient