Protein NMR Spectroscopy at 150 kHz Magic‐Angle Spinning Continues To Improve Resolution and Mass Sensitivity

Abstract

Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic‐angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.

Temperature-Dependent Solid-State NMR Proton Chemical-Shift Values and Hydrogen Bonding

“…The reported temperature dependence of proton chemical-shift values for amide protons are small, in the order of a few parts per billion per Kelvin (ppb/K), and thus require well-resolved proton resonances, which can typically be achieved by performing the NMR experiments at >100 kHz MAS. [28][29][30] An additional technical complication arises from the finding that a change of the sample temperature is accompanied by temperature changes in the body of the probe and the bore of the magnet causing a change in the magnetic susceptibility and thus of the magnetic field at the sample position ( �⃗ 0,sample ). Inserting the sample into the probe at the beginning of the experiment, sample change or even a sample-temperature change leads to a new temperature distribution in the entire probe and, by thermal contact, also within the bore of the magnet.…”

Protein Side-Chain–DNA Contacts Probed by Fast Magic-Angle Spinning NMR

“… 24 , 40 , 60 , 61 Those fwhm are still dominated by homogeneous line broadening mechanisms which could potentially be further scaled down by faster MAS experiments. 60 , 62 , 63 …”

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

“…While 13 C chemical shift anisotropies are, for instance, on the order of a few kHz, and already very low spinning frequencies are sufficient to average them, it is highly beneficial to spin even faster than 100 kHz to average the strong proton–proton dipolar interactions. These approaches are currently under development [ 28 , 29 ].…”

“…Still, the solid-state linewidth is typically somewhat broader than in solution-state NMR, which results in a higher spectral crowding than in solution, and limits the protein size to be investigated. Thus, further improvements in MAS frequency (>100 kHz [ 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]) and magnetic field strength (>800 MHz or 18.8 Tesla; note that magnetic field strength is conventionally expressed in units of the proton resonance frequency) will be important for the study of larger proteins. Typically, going from 800 MHz to 1.2 GHz increases the signal-to-noise ratio by nearly a factor of 2.…”

“…Until recently, solid-state NMR samples routinely comprised >20 mg of pure protein. With the advent of advanced proton-detection fast MAS techniques, proteins can now be analyzed in 0.7 mm and even 0.5 mm rotors which reach over 150 kHz MAS frequencies ( Figure 1 a), yielding well-resolved proton-nitrogen correlation spectra [ 29 , 34 , 35 , 36 , 53 ]. The concomitant reduction in sample sizes to the sub-milligram level enables the use of alternative production techniques to bacterial expression.…”