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Protein Backbone and Side Chain Assignment using Proton Detection under Ultra-Fast Magic Angle Spinning

Since recent Bruker BioSpin developments have seen a move towards smaller rotor sizes down to 0.7 mm, the use of MAS rates up to 111 kHz became possible, allowing for 1H-detected solid-state NMR of biological samples. Based on the previously announced Application Note “Introduction to Proton Detection in Biological Samples under Ultra-Fast Magic Angle Spinning” (download here), Bruker BioSpin now presents a full set of ten 1H-detected 1D to 3D experiments for an efficient resonance assignment of a protein backbone and its amino acid side chains. Since the introduced experiments yield either correlations within one amino acid or between consecutive residues, especially the combined analysis of intra- and interresidue correlation spectra, reconciled with the protein amino acid sequence, allows for a fast ‘sequential walk’ assignment.

In the ultra-fast magic-angle spinning (MAS) regime of 60 to 111 kHz, strong unwanted 1H-dipolar couplings are sufficiently attenuated to resolve 1H-detected signals derived from a protein sample. As a consequence of this MAS-induced decoupling, polarization transfer using scalar couplings becomes efficient. Therefore, in all relevant experiments presented here, homonuclear 13C-13C polarization transfer is mediated through-bond by the scalar-based INEPT, a well-known scheme in solution NMR spectroscopy. Typical fast transverse relaxation of Cα spins and the unwanted signal loss to potential third spins (such as Cβ) is omitted by the use of the out-and-back transfer scheme, which is derived from the field of solution NMR as well.

Similar to the previous Application Note, a detailed description about these introduced schemes and a summary of parameter recommendations is depicted. In addition, a straight-forward optimization protocol guarantees a smooth and easy workflow. A flowchart directs the user step by step through the optimization process of all relevant parameter settings, which is done in one-dimensional experiments only. Because of the unambiguous parameter convention, each optimized parameter can be transferred easily to subsequent experiments.

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