Immobilization of soluble protein complexes in MAS solid-state NMR: Sedimentation versus viscosity

Abstract

In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained.

Revealing Intermolecular Hydrogen Bonding Structure and Dynamics in a Deep Eutectic Pharmaceutical by Magic-Angle Spinning NMR Spectroscopy

“…While CP is efficient at low temperatures in “solid-like” samples, the efficacy decays upon heating as dipolar couplings are partially averaged by molecular motion. Our observation matches that of Sarkar et al, who reported CP transfers in a high viscosity natural DES (glucose/choline chloride/water in a 1:1:4 ratio) with increasing transfer efficiency upon a decrease in temperature . Overall, the result is a region between liquid and solid phases (between 268 and 288 K for the case of Lid·Ibu) in which the transfer of coherences from the 1 H to 13 C by either technique is inefficient.…”

“…Our observation matches that of Sarkar et al, who reported CP transfers in a high viscosity natural DES (glucose/choline chloride/water in a 1:1:4 ratio) with increasing transfer efficiency upon a decrease in temperature. 24 Overall, the result is a region between liquid and solid phases (between 268 and 288 K for the case of Lid•Ibu) in which the transfer of coherences from the 1 H to 13 C by either technique is inefficient. Inspection of the 1D proton spectra as a function of temperature reveals that while the chemical shifts of the CH protons are essentially unchanged, there are changes for the NH and COOH protons, which can be attributed to different interactions and chemical exchange processes.…”

Structural Biology of Iron‐Binding Proteins by NMR Spectroscopy

“…This activity on ferritin was valuable for providing the experimental evidence for correctly locating the ferroxidase site and emerging iron‐cluster precursors of the biomineral, albeit in a low‐resolution mode. On the other hand, at CERM ferritin is used as a model system for testing new pulse sequences for very large proteins; the most relevant results are related to the development of the so‐called SedNMR (i.e., sedimented solute NMR) approach, where pelleted proteins, obtained by ultracentrifugation or by direct spinning inside the MAS rotor concentrated protein solutions, are analyzed via solid state MAS NMR spectroscopy …”

Chemical shift-based methods in NMR structure determination

“…Ever since the first de novo atomic-resolution structure determined by NMR in 1985 [238], chemical shifts have remained an invaluable tool for spectroscopists towards examining the structure and dynamics of biomolecules for systems up to 1 MDa (Megadalton) in the solution- [174] and solid-state [239]. More recently, NMR methods have been applied to determine protein structures within living cells [240].…”