Characterization of Alzheimer's-like paired helical filaments from the core domain of tau protein using solid-state NMR spectroscopy

The polymerization of the microtubule-associated protein tau into paired helical filaments (PHFs) represents one of the hallmarks of Alzheimer's disease. We employed solid-state nuclear magnetic resonance (NMR) to investigate the structure and dynamics of PHFs formed in vitro by the three-repeat-domain (K19) of protein tau, representing the core of Alzheimer PHFs. While N and C termini of tau monomers in PHFs are highly dynamic and solvent-exposed, the rigid segment consists of three major beta-strands. Combination of through-bond and through-space ssNMR transfer methods with water-edited ((15)N, (13)C) and ((13)C, (13)C) correlation experiments suggests the existence of a fibril core that is largely built by repeat unit R3, flanked by surface-exposed units R1 and R4. Solid-state NMR, circular dichroism, and the fibrillization behavior of a K19 mutant furthermore indicate that electrostatic interactions play a central role in stabilizing the K19 PHFs.     

Solid-state NMR adiabatic TOBSY sequences provide enhanced sensitivity for multidimensional high-resolution magic-angle-spinning 1H MR spectroscopy

We propose a solid-state NMR method that maximizes the advantages of high-resolution magic-angle-spinning (HRMAS) applied to intact biopsies when compared to more conventional liquid-state NMR approaches. Theoretical treatment, numerical simulations and experimental results on intact human brain biopsies are presented. Experimentally, it is proven that an optimized adiabatic TOBSY (TOtal through Bond correlation SpectroscopY) solid-state NMR pulse sequence for two-dimensional 1H-1H homonuclear scalar-coupling longitudinal isotropic mixing provides a 20%-50% improvement in signal-to-noise ratio relative to its liquid-state analogue TOCSY (TOtal Correlation SpectroscopY). For this purpose we have refined the C9(15)1 symmetry-based 13C TOBSY pulse sequence for 1H MRS use and compared it to MLEV-16 TOCSY sequence. Both sequences were rotor-synchronized and implemented using WURST-8 adiabatic inversion pulses. As discussed theoretically and shown in simulations, the improved magnetization-transfer comes from actively removing residual dipolar couplings from the average Hamiltonian. Importantly, the solid-state NMR techniques are tailored to perform measurements at low temperatures where sample degradation is reduced. This is the first demonstration of such a concept for HRMAS metabolic profiling of disease processes, including cancer, from biopsies requiring reduced sample degradation for further genomic analysis.     

Probing structure in the polymorphic domain of the L-enantiomer of N-benzoyl-phenylalanine by means of 2D solid-state NMR spectroscopy and DFT calculations

A study of polymorphism using a range of solid-state NMR techniques is presented. We demonstrate the existence of at least six polymorphs in a sample of N-benzoyl-L-phenylalanine. We also present methodology for the characterization of the protonation state, hydrogen bonding, and molecular conformation for the polymorphs, together with results of such a characterization for one of the polymorphs present in our sample. DFT modeling is used to investigate the separate effects hydrogen bonding and molecular conformation have on the chemical shift tensor.     

Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.