Reconstitution of Isotopically Labeled Ribosomal Protein L29 in the 50S Large Ribosomal Subunit for Solution-State and Solid-State NMR

Abstract

Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S ribosomal subunit. In particular, we focus on the 63-residue ribosomal protein L29 (~7 kDa), which is located at the exit of the tunnel of the large 50S ribosomal subunit (~1.5 MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP).

Deuteration for High-Resolution Detection of Protons in Protein Magic Angle Spinning (MAS) Solid-State NMR

“…In a typical MAS solid-state NMR experiment, rotation of the sample induces accelerations due to centrifugation on the order of 1–10 × 10 6 g. , Depending on its molecular weight, sedimentation efficiently immobilizes the soluble protein. Essential for the success of the method is the development of packing tools that allow efficient transfer of protein material into the solid-state NMR rotor. − Using this approach, the 20S proteasome, the 50S ribosome, , the 50S bound trigger factor, and immunoglobulins in complex with GB1 have been investigated. Small heat shock proteins such as αB-crystallin assemble into oligomers with a molecular weight of 400–600 kDa.…”

“…Similarly, on average, a MAS frequency of (41 ± 28) kHz is necessary to achieve the same intensity for CH 2 D versus CHD 2 selectively methyl protonated samples. This information is potentially of interest for experiments that involve very large, nonsymmetric protein complexes where signal-to-noise is critical and for which any additional increase in sensitivity is essential for the success of the experiment. ,− …”

Field and magic angle spinning frequency dependence of proton resonances in rotating solids

“…This information is potentially of interest for the design of experiments involving very large protein complexes for which sensitivity is limiting, and for which an additional gain in sensitivity would be desirable [30,[85][86][87].…”

An Efficient, Robust New Scheme for Establishing Broadband Homonuclear Correlations in Biomolecular Solid State NMR

“…Continuous technological and conceptual developments have endowed contemporary high‐resolution solid‐state nuclear magnetic resonance (NMR) spectroscopy, with capabilities that rival those of solution‐state NMR when tackling structural and dynamic investigations of large biomolecules . When performing such studies the problem of spectral assignment and resolution nearly invariably arises, demanding the use of high‐dimensional NMR experiments correlating labeled 13 C‐ and/or 15 N sites in the biomolecule.…”

“…Continuous technological and conceptual developments have endowed contemporary high-resolution solid-state nuclear magnetic resonance (NMR) spectroscopy, with capabilities that rival those of solution-state NMR when tackling structural and dynamic investigations of large biomolecules. [1][2][3][4][5][6][7][8] When performing such studies the problem of spectral assignment and resolution nearly invariably arises, demanding the use of highdimensional NMR experiments correlating labeled 13 C-and/or 15 N sites in the biomolecule. This in turn often calls for reintroducing the dipolar interactions among these labeled nuclei, that are otherwise averaged out by the magic-anglespinning (MAS) procedure required for achieving site-specific high-resolution information in this kind of study.…”