Multidimensional Magic Angle Spinning NMR Spectroscopy for Site-Resolved Measurement of Proton Chemical Shift Anisotropy in Biological Solids

Abstract

The proton chemical shift (CS) tensor is a sensitive probe of structure and hydrogen bonding. Highly accurate quantum-chemical protocols exist for computation of 1H magnetic shieldings in the various contexts, making proton chemical shifts potentially a powerful predictor of structural and electronic properties. However, 1H CS tensors are not yet widely used in protein structure calculation due to scarcity of experimental data. While isotropic proton shifts can be readily measured in proteins even in the solid state, determination of the 1H chemical shift anisotropy (CSA) tensors remains challenging, particularly in molecules containing multiple proton sites. We present a method for site-resolved measurement of amide proton CSAs in fully protonated solids under magic angle spinning. The approach consists of three concomitant 3D experiments yielding spectra determined by either mainly 1H CSA, mainly 1H-15N dipolar, or combined 1H CSA and 1H-15N dipolar interactions. The anisotropic interactions are recoupled using RN-sequences of appropriate symmetry, such as , and 15N/13C isotropic CS dimensions are introduced via a short selective 1H-15N cross-polarization step. Accurate 1H chemical shift tensor parameters are extracted by simultaneous fit of the lineshapes recorded in the three spectra. An application of this method is presented for an 89-residue protein, U-13C,15N-CAP-Gly domain of dynactin. The CSA parameters determined from the triple fits correlate with the hydrogen-bonding distances, and the trends are in excellent agreement with the prior solution NMR results. This approach is generally suited for recording proton CSA parameters in various biological and organic systems, including protein assemblies and nucleic acids.

Measuring proton shift tensors with ultrafast MAS NMR

“…Note that in the case of OH protons, resolution of different sites cannot be improved by magnetization transfer to a directly bonded heteronucleus as in Ref. [19].…”

“…Suitable symmetries must also avoid inadvertently recoupling the strong 1 H dipolar interactions, as well as isotropic 1 H shifts. Of the many symmetries which satisfy these conditions, R18 2 5 [12] and R12 1 4 [19] have been used successfully to measure 1 H shift tensor parameters.…”

“…For R18 2 5 and R12 1 4 rf amplitudes in excess of 180 kHz and 240 kHz, respectively, would be required for MAS rates above 40 kHz, and in Refs. [12] and [19] experiments were carried out below 20 kHz. A different choice of sequence symmetry allows similar experiments to be carried out at higher MAS rates at which a well-resolved isotropic 1 H spectrum can be acquired without the need for homonuclear decoupling as in Ref.…”

“…In common with all methods based on rotary resonance, the latter suffers from substantial sensitivity to rf inhomogeneity, while the sequence symmetry chosen in the former restricts the experiment to relatively slow MAS rates. In order to improve the resolution of different sites in ν2, Hou et al [19] recently exploited magnetization transfer to a neighbouring heteronucleus after recoupling the 1 H shift anisotropy during t1. This approach was shown to be particularly powerful for measuring the 1 H shift tensor parameters for the amide protons in 15 N-enriched proteins, but is less appropriate for hydrogen-bonded OH sites where a suitable directly bonded heteronucleus is not available.…”

1 H CSA parameters by ultrafast MAS NMR: Measurement and applications to structure refinement

“…For example, Brouwer and Ripmeester [1] resolved 1 H isotropic shifts in ν2 by a combination of moderate MAS (ωr/2π = 16 kHz) and multi-pulse homonuclear decoupling and used a recoupling sequence designed using symmetry principles [2,3] during t1. Subsequently, Hou et al [4] optimized the resolution of 1 H sites by magnetization transfer to a neighbouring 15 N nucleus after recoupling the 1 H CSA in a similar fashion and measured the 1 H shift parameters for the amide sites in 15 N-enriched proteins. Miah et al [5] employed modified recoupling sequences suitable for use with "ultrafast" MAS (ωr/2π > 50 kHz), an approach that allows hydrogenbonded sites in simple crystalline solids to be resolved in ν2 without the need for multi-pulse homonuclear decoupling.…”