Advances in instrumentation and methodology for solid-state NMR of biological assemblies

Abstract

Many advances in instrumentation and methodology have furthered the use of solid-state NMR as a technique for determining the structures and studying the dynamics of molecules involved in complex biological assemblies. Solid-state NMR does not require large crystals, has no inherent size limit, and with appropriate isotopic labeling schemes, supports solving one component of a complex assembly at a time. It is complementary to cryo-EM, in that it provides local, atomic-level detail that can be modeled into larger-scale structures. This review focuses on the development of high-field MAS instrumentation and methodology; including probe design, benchmarking strategies, labeling schemes, and experiments that enable the use of quadrupolar nuclei in biomolecular NMR. Current challenges facing solid-state NMR of biological assemblies and new directions in this dynamic research area are also discussed.

Keywords: Biological assemblies; Instrumentation; MAS probe; Magic angle spinning; Solid-state NMR.

Figure 1:

A comparision of the different probe circuit styles discussed above, using representative examples from the literature. Shown are circuit diagrams and images of a lumped element probe (a-b) [63], a tuning tube probe (c-d) [62], and a transmission line probe (e-f) [64].

Figure 2:

A comparision of the 810°/90° ratios for the two crossed coils in a quadruple-resonance MAS probe [62]. (a) The ¹H nutation on the MAG coil, and (b) the ¹⁵N nutation on the variable pitch solenoid.

Figure 3:

Methods for assessing the rf homogeneity of an NMR probe (a-b) Magnitude profiles for the B₁ field at two frequencies calculated for two positions within a 3.2 mm rotor, at the center of the rotor (a) and along the inner wall of the rotor (b) in a solenoid coil [77]; (c-e) NMR measurements of the rf field profile (c) shows the locations of the silicone spacers in the rotor, (d) shows the 1D ¹H spectra of the silicone spacer at each location, and (e) is the 2D ¹H nutation spectra of the same [74]; f) CAD image of a tool used to perform ball-shift measurements [90]; g) homogeneity measurements taken using the tool in (f) [62]; h) nutation curve of the ²H channel of a quadruple-resonance MAS probe [62]; i-j) 2D ¹H nutation spectra of adamantane (at 400 MHz), (j) is an expansion of the center part of the spectrum in (i) [83].

Figure 4:

Crossed coil assemblies and transverse coils that can be used in such; a) the original Alderman-Grant resonator [69], b) the Zhang MAG [123], c) the Gor’kov MAS crossed coil assembly [63], d) the Martin lab MAS crossed coil assembly [62] using a version the Opella lab’s compact MAG design [122], e) the Doty XC on a ceramic support (the crossed coil assembly would have a variable-pitch solenoid wound around this) [65].

Figure 5:

a) The Sakellariou MACS microcoil fits inside the MAS rotor [124], b) The doubly-tuned microcoil of Takeda et al enables double-resonance MAS [127], c) The Kent-gens piggyback coil enhances the signal in small samples [128].

Figure 6:

¹⁷O spectra demonstrating the utility of this nucleus for characterizing reaction mechanisms, particularly in acid-base chemistry. Here, the binding of a substrate to the enzyme tryptophan synthase is observed [150].

Figure 7:

Spectral resolution for a microcrystalline sample of the protein GB1 at 60 kHz MAS can be improved by deuteration, while at 111 kHz MAS the difference between the spectra of deuterated and fully protonated GB1 is almost negligible [156].

Figure 8:

Panels A, B, and C show three different orientations of GB1. The color coding indicates di erences in overall mobility based on site-specific evaluation of 140 aliphatic sidechain sites using deuterium labeling. Red, orange and blue indicate the amplitude of motion, being large, moderate and small, respectively [172].

Figure 9:

A) 3D spectrum of labeled ubiquitin from ²H_DQ-¹³C-¹³ correlation experiments. B) 2D slices can be used to determine connectivities and assign resonances similar to conventional solution-state techniques. The resolution achieved was also suitable for studies on a much larger membrane protein, OmpG. [173].

Figure 10:

Above is an illustration of observed chemical shift perturbations as they relate to the overall structure upon binding of a ligand, UCB-FcRn-303, to the extracellular domain of a 42 kDa, fully protonated neonatal Fc receptor (FcRn). This receptor was chosen because it has been established as a drug target for several autoimmune diseases. Color denotes the magnitude of the perturbation as shown by the scale [188].