Solid State NMR at CERM/CIRMMP in Florence, Italy

Solid-state NMR methods have the unique capability of providing detailed structural constraints for amyloid fibrils for the development of full molecular models, conformational dynamics, and fibrils assembly pathways at an atomic level. Solid-state NMR has been applied to structurally investigate disease-related problems, such as fibrils associated with Alzheimer's disease, prion fibrils, and related systems. Solid-state NMR techniques have been applied and are available at the infrastructure for the determination of fibril structures. Detailed structural studies can be accomplished by exploitation of the effect induced by the presence of paramagnetic metal ions. When applicable, crystallization can take advantage of the robotic system.

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User Guide

CERM/CIRMMP has experience with several different kinds of biosolids:

-Microcrystalline proteins

-Amyloids and fibrils

-Sedimented solutes

-Paramagnetic systems

CERM/CIRMMP has a long tradition in the development of new NMR methods: 1) determination of paramagnetic effects in the solid state to access structural information, 2) 13C experiments with homonuclear decoupling in the direct dimension through spin-state-selection, 3) 1H direct detection. Users can thus perform the following experiments:

1H detected experiments – (13C, 15N, 2H labeled samples, back exchanged in H2O)

-Assignment, determination of structural constraints, local dynamics

-Determination of paramagnetic effects

13C detected experiments – (13C, 15N labeled samples)

-Assignment, determination of structural constraints, local dynamics

-Determination of paramagnetic effects

Solid State NMR spectroscopy measurements can be performed in virtually any kind of biological solid. 13C-15N labelled samples are required, since the vast majority of the experiments are based on direct heteronuclear detection. Recently, 2H labelled proteins back exchanged in water are becoming popular, since they allow for proton detection. Local order in the solid as well as its hydration state are known to dramatically affect spectral quality; micro- to nanocrystalline proteins are therefore the target of these studies.

Magic angle spinning is used to average the anisotropic interactions, so the sample is packed in zirconia rotors from 1.3 to 4 mm. The amount of sample ranges from about 2 mg in the 1.3 rotor to 50 mg in the 4 mm rotor. Despite the much smaller amount of material, the signal to noise ratio in the 1.3 rotor can be almost as good as in larger rotors, because, among other factors, of the higher efficiency of the smaller coils. Since the RF heating in SSNMR is more severe than in solution, low salt samples are required.

The acquisition of spectra takes from a few minutes (1D spectra) to a few days (3D spectra), depending on the amount of sample and its quality.

Once the sample is placed in the probehead and the desired temperature and spinning rates have been reached, the pulses are calibrated and polarization transfer (from proton to insensitive nuclei) must be set up. Usually it is accomplished by cross-polarization, while in highly mobile systems it may be obtained by INEPT, as in liquids. Several pulse sequences are routinely available for assignment and structural characterization, and local and global dynamics may be easily estimated.

The spectrometer provides time-domain data that are transformed automatically by the software into frequency-domain data.