Probing the Influence of Single-Site Mutations in the Central Cross-β Region of Amyloid β (1–40) Peptides

Amyloid β (Aβ) is a peptide known to form amyloid fibrils in the brain of patients suffering from Alzheimer’s disease. A complete mechanistic understanding how Aβ peptides form neurotoxic assemblies and how they kill neurons has not yet been achieved. Previous analysis of various Aβ40 mutants could reveal the significant importance of the hydrophobic contact between the residues Phe19 and Leu34 for cell toxicity. For some mutations at Phe19, toxicity was completely abolished. In the current study, we assessed if perturbations introduced by mutations in the direct proximity of the Phe19/Leu34 contact would have similar relevance for the fibrillation kinetics, structure, dynamics and toxicity of the Aβ assemblies. To this end, we rationally modified positions Phe20 or Gly33. A small library of Aβ40 peptides with Phe20 mutated to Lys, Tyr or the non-proteinogenic cyclohexylalanine (Cha) or Gly33 mutated to Ala was synthesized. We used electron microscopy, circular dichroism, X-ray diffraction, solid-state NMR spectroscopy, ThT fluorescence and MTT cell toxicity assays to comprehensively investigate the physicochemical properties of the Aβ fibrils formed by the modified peptides as well as toxicity to a neuronal cell line. Single mutations of either Phe20 or Gly33 led to relatively drastic alterations in the Aβ fibrillation kinetics but left the global, as well as the local structure, of the fibrils largely unchanged. Furthermore, the introduced perturbations caused a severe decrease or loss of cell toxicity compared to wildtype Aβ40. We suggest that perturbations at position Phe20 and Gly33 affect the fibrillation pathway of Aβ40 and, thereby, influence the especially toxic oligomeric species manifesting so that the region around the Phe19/Leu34 hydrophobic contact provides a promising site for the design of small molecules interfering with the Aβ fibrillation pathway

Multiple-quantum magic-angle spinning: high-resolution solid state NMR spectroscopy of half-integer quadrupolar nuclei

Summary. Experimental and theoretical aspects of the multiple-quantum magic-angle spinning experiment (MQMAS) are discussed in this review. The significance of this experiment, introduced by Frydman and Harwood, is in its ability to provide high-resolution NMR spectra of half-integer quadrupolar nuclei (I greater than or equal to 3/2). This technique has proved to be useful in various systems ranging from inorganic materials to biological samples. This review addresses the development of various pulse schemes aimed at improving the signal-to-noise ratio and anisotropic lineshapes. Representative spectra are shown to underscore the importance and applications of the MQMAS experiment

NMR Crystallography at Fast Magic-Angle Spinning Frequencies: Application of Novel Recoupling Methods

Chemical characterisation of active pharmaceutical compounds can be challenging, especially when these molecules exhibit tautomeric or desmotropic behaviour. The complexity can increase manyfold if these molecules are not susceptible to crystallisation. Solid-state NMR has been employed effectively for characterising such molecules. However, characterisation of a molecule is just a first step in identifying the differences in the crystalline structure. 1 H solid-state Nuclear Magnetic Resonance (ssNMR) studies on these molecules at fast magic-angle-spinning frequencies can provide a wealth of information and may be used along with ab initio calculations to predict the crystal structure in the absence of X-ray crystallographic studies. In this work, we attempted to use solid-state NMR to measure 1 H - 1 H distances that can be used as restraints for crystal structure calculations. We performed studies on the desmotropic forms of albendazole

Build-up curves.

<p>Magnetisation build-up curves obtained using (a) PARIS (N = 1/2), (b) PARIS-xy (m = 1)(N = 1/2), and (c) PARIS (N = 2) recoupling schemes for different cross-peaks of U-¹³C-,¹⁵N-L-histidineHO, at  = 30 kHz and  = 11.74 T. Each recoupling scheme was applied for a fixed mixing-time period (90 ms) using different RF amplitudes of 7.3, 16, 24, 30, and 40 kHz. values calculated for individual cross-peaks according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050504#pone.0050504.e052" target="_blank">Eq. 1</a> are plotted as a function of RF amplitude. All values are normalised with respect to the maximum value yielded by PARIS (N = 2) using a RF amplitude of 40 kHz. Data points are joined with straight lines to guide the readers' eyes.</p

Schematic of recoupling pulse sequence and blocks.

<p>(a) General pulse scheme for a 2D ¹³C-¹³C spin-diffusion experiment. Second-order recoupling schemes (b) DARR, (c) PARIS (N = 1/2), (d) PARIS (N = 2), (e) PARIS-xy (m = 1)(N = 1/2), (f) PARIS-xy (m = 1)(N = 2), and (g) PARIS-xy (M = 2)(N = 1/2) are applied on the ¹H channel during the mixing period.</p

Build-up curves.

<p>Magnetisation build-up curves of (a) C -C , (b) C -C , (c) C -C , (d) C -C , (e) C -CO, (f) C -CO, (g) C -CO, and (h) C -C cross-peaks of U-¹³C-,¹⁵N-L-histidineHO for different recoupling schemes applied on the ¹H channel, at  = 30 kHz and  = 16.43 T. Each recoupling scheme was applied for a fixed mixing-time period (90 ms) using different RF amplitudes of 7.3, 16, 24, 30, and 40 kHz. values calculated for individual cross-peaks according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050504#pone.0050504.e052" target="_blank">Eq. 1</a> are plotted as a function of RF amplitude. values are normalised with respect to the maximum value yielded by PARIS (N = 1/2) ( = 16 kHz) in (a, b, and c), PARIS-xy (m = 1)(N = 2) ( = 30 kHz) in (d), PARIS (N = 2) ( = 30 kHz) in (e and f), PARIS-xy (m = 2)(N = 1/2) ( = 30 kHz) in (g), and PARIS (N = 1/2) ( = 40 kHz) in (h). Data points are joined with straight lines to guide the readers' eyes.</p

¹³C-¹³C spectra.

<p>2D ¹³C-¹³C spectra of U-¹³C-,¹⁵N-fMLF recorded with (a and b) PARIS (N = 1/2), (c and d) PARIS (N = 1/2), (e and f) (e) PARIS-xy (m = 1)(N = 1/2), (g and h) PARIS-xy (m = 1)(N = 2), (i and j) PARIS-xy (M = 2)(N = 1/2), (k) DARR, and (l) PDSD recoupling scheme using an RF amplitude of 16 kHz in (a, c, e, g, and i), 30 kHz in (b, d, f, h, j, and k) and without RF in (l). Each spectrum was recorded at a MAS frequency of 30 kHz, magnetic field of 16.43 T, and using a 90 ms long mixing-time period.</p

2D ¹³C-¹³C spin-diffusion spectrum.

<p>(a) A schematic molecular structure of histidine molecule. A map of all possible cross-peaks in a 2D ¹³C-¹³C spin-diffusion spectrum of U-¹³C-,¹⁵N-L-histidineHO as a function of chemical-shift difference (along X axis) and spatial distance (along Y axis) between two carbon atoms at magnetic fields of (b) 11.74 and (c) 16.43 T. Highlighted cross-peaks are the ones which are studied in this work.</p

¹³C-¹³C spectra.

<p>2D ¹³C-¹³C spectra of U-¹³C-,¹⁵N-fMLF recorded using PARIS-xy (m = 1)(N = 1/2) recoupling scheme at MAS frequency of 30 kHz and an external magnetic field of 16.43 T. Mixing-time period in each case was 90 ms. Different proton RF amplitudes of (a) 7.3 kHz, (b) 16 kHz, (c) 24 kHz, and (d) 30 kHz were employed. Only a selected region of 2D spectrum, covering cross-peaks from each class, is shown in each case.</p

¹³C-¹³C spectra.

<p>2D ¹³C-¹³C spectra of A recorded at  = 16.43 T and  = 30 kHz. PARIS-xy (m = 1)(N = 1/2) recoupling scheme was employed during a 90 ms long mixing-time period using RF amplitudes of (a, b, c) 16 and (d, e, f) 30 kHz. Selected regions of 2D spectrum, covering (a, d) aliphatic-carbonyl, (b, e) aliphatic-aromatic, and (c, f) aliphatic-aliphatic cross-peaks, are shown in both the cases.</p