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Proteins often undergo large-scale conformational transitions, in which secondary and tertiary structure elements (loops, helices, and domains) change their structures or their positions with respect to each other. Simple considerations suggest that such dynamics should be relatively fast, but the functional cycles of many proteins are often relatively slow. Sophisticated experimental methods are starting to tackle this dichotomy and shed light on the contribution of large-scale conformational dynamics to protein function. In this review, we focus on the contribution of single-molecule Förster resonance energy transfer and nuclear magnetic resonance (NMR) spectroscopies to the study of conformational dynamics. We briefly describe the state of the art in each of these techniques and then point out their similarities and differences, as well as the relative strengths and weaknesses of each. Several case studies, in which the connection between fast conformational dynamics and slower function has been demonstrated, are then introduced and discussed. These examples include both enzymes and large protein machines, some of which have been studied by both NMR and fluorescence spectroscopies. [ABSTRACT FROM AUTHOR]

Dynamic processes in molecules can occur on a large range of time scales, and it is important to understand which time scales of motion contribute to different parameters used in dynamics measurements. For spin relaxation, this can easily be understood from the sampling of the spectral-density function by different relaxation-rate constants. In addition to data from relaxation measurements, determining dynamically-averaged anisotropic interactions in magic-angle spinning (MAS) solid-state NMR allows better quantification of the amplitude of molecular motion. For partially averaged anisotropic interactions, the relevant time scales of motion are not so clearly defined and whether the averaging depends on the experimental methods (e.g., pulse sequences) or conditions (e.g., MAS frequency, magnitude of anisotropic interaction, rf-field amplitudes) is not fully understood. To investigate these questions, we performed numerical simulations of dynamic systems based on the stochastic Liouville equation using several experiments for recoupling the dipolar-coupling, CSA or quadrupolar coupling. The transition between slow motion, where parameters characterizing the anisotropic interaction are not averaged, and fast motion, where the tensors are averaged leading to a scaled anisotropic quantity, occurs over a window of motional rate constants that depends mainly on the strength of the interaction. This transition region can span two orders of magnitude in exchange-rate constants (typically in the μs range) but depends only marginally on the employed recoupling scheme or sample spinning frequency. Residual couplings in off-magic-angle experiments, however, average over longer time scales of motion. While in principle one may gain information on the time scales of motion from the transition area, extracting such information is hampered by low signal-to-noise ratio in experimental spectra due to fast relaxation that occurs in the same region. [ABSTRACT FROM AUTHOR]

Amide-proton detected magic-angle spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D2O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with H2O-based culture medium doped with deuterated cell lysate allow to overcome this 'reprotonation bottleneck', while retaining a high level of deuteration (ca. 80 %) and narrow line widths. We quantified coherence life times of several proteins prepared with this labelling pattern over a range of MAS frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), amide 1H line widths with our labelling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and show that such media can be used identically to commercial media. The residual protonation of Hα sites allows for well-resolved Hα-detected spectra and Hα resonance assignment, exemplified by the de novo assignment of 168 Hα sites in a 39 kDa protein. The approach based on this H2O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm/100 kHz MAS experiments. [ABSTRACT FROM AUTHOR]

Amide-proton-detected magic-angle-spinning NMR of deuterated proteins has become a main technique in NMR-based structural biology. In standard deuteration protocols that rely on D 2 O-based culture media, non-exchangeable amide sites remain deuterated, making these sites unobservable. Here we demonstrate that proteins produced with a H 2 O-based culture medium doped with deuterated cell lysate allow scientists to overcome this "reprotonation bottleneck" while retaining a high level of deuteration (ca. 80 %) and narrow linewidths. We quantified coherence lifetimes of several proteins prepared with this labeling pattern over a range of magic-angle-spinning (MAS) frequencies (40–100 kHz). We demonstrate that under commonly used conditions (50–60 kHz MAS), the amide 1 H linewidths with our labeling approach are comparable to those of perdeuterated proteins and better than those of protonated samples at 100 kHz. For three proteins in the 33–50 kDa size range, many previously unobserved amides become visible. We report how to prepare the deuterated cell lysate for our approach from fractions of perdeuterated cultures which are usually discarded, and we show that such media can be used identically to commercial media. The residual protonation of H α sites allows for well-resolved H α -detected spectra and H α resonance assignment, exemplified by the de novo assignment of 168 H α sites in a 39 kDa protein. The approach based on this H 2 O/cell-lysate deuteration and MAS frequencies compatible with 1.3 or 1.9 mm rotors presents a strong sensitivity benefit over 0.7 mm 100 kHz MAS experiments. [ABSTRACT FROM AUTHOR]

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Aromatic Side Chains, Isotopic Labeling, Protein Dynamics, Ring Flips, Spin Relaxation Keywords: Aromatic Side Chains; Isotopic Labeling; Protein Dynamics; Ring Flips; Spin Relaxation EN Aromatic Side Chains Isotopic Labeling Protein Dynamics Ring Flips Spin Relaxation 1 1 1 04/26/23 20230502 NES 230502 B Rock-solid amyloid? Cover Picture: The Rigid Core and Flexible Surface of Amyloid Fibrils Probed by Magic-Angle-Spinning NMR Spectroscopy of Aromatic Residues (Angew. [Extracted from the article]

These conclusions could be drawn from dynamics measurements by solid-state MAS NMR in combination with specific labeling of phenylalanines and tyrosines as Paul Schanda et al. describe in their Research Article (e202219314). Aromatic residues in the core of the amyloid fibrils HELLF and HET-s show no sign of ring flips suggesting a rigid core that does not undergo "breathing motions". These conclusions could be drawn from dynamics measurements by solid-state MAS NMR in combination with specific labeling of phenylalanines and tyrosines as Paul Schanda et al. describe in their Research Article (e202219314). [Extracted from the article]

Holdase chaperones are essential in the mitochondrial membrane-protein biogenesis as they stabilize preproteins and keep them in an import-competent state as they travel through the aqueous cytosol and intermembrane space. The small TIM chaperones of the mitochondrial intermembrane space function within a fine balance of client promiscuity and high affinity binding, while being also able to release their client proteins without significant energy barrier to the downstream insertases/translocases. The tendency of the preproteins to aggregate and the dynamic nature of the preprotein-chaperone complexes makes the preparation of these complexes challenging. Here we present two optimized methods for complex formation of highly hydrophobic precursor proteins and chaperones: a pull-down approach and an in-vitro translation strategy. In the former, attaching the client protein to an affinity resin keeps the individual client protein copies apart from each other and decreases the client self-aggregation probability, thereby favouring complex formation. In the latter approach, a purified chaperone, added to the cell-free protein synthesis, captures the nascent precursor protein. The choice of method will depend on the desired client-chaperone complex amount, or the need for specific labeling scheme., (Copyright © 2024. Published by Elsevier Inc.)

Aromatic side chains are important reporters of the plasticity of proteins, and often form important contacts in protein-protein interactions. We studied aromatic residues in the two structurally homologous cross-β amyloid fibrils HET-s, and HELLF by employing a specific isotope-labeling approach and magic-angle-spinning NMR. The dynamic behavior of the aromatic residues Phe and Tyr indicates that the hydrophobic amyloid core is rigid, without any sign of "breathing motions" over hundreds of milliseconds at least. Aromatic residues exposed at the fibril surface have a rigid ring axis but undergo ring flips on a variety of time scales from nanoseconds to microseconds. Our approach provides direct insight into hydrophobic-core motions, enabling a better evaluation of the conformational heterogeneity generated from an NMR structural ensemble of such amyloid cross-β architecture., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Probing the dynamics of aromatic side chains provides important insights into the behavior of a protein because flips of aromatic rings in a protein's hydrophobic core report on breathing motion involving a large part of the protein. Inherently invisible to crystallography, aromatic motions have been primarily studied by solution NMR. The question how packing of proteins in crystals affects ring flips has, thus, remained largely unexplored. Here we apply magic-angle spinning NMR, advanced phenylalanine 1 H- 13 C/ 2 H isotope labeling and MD simulation to a protein in three different crystal packing environments to shed light onto possible impact of packing on ring flips. The flips of the two Phe residues in ubiquitin, both surface exposed, appear remarkably conserved in the different crystal forms, even though the intermolecular packing is quite different: Phe4 flips on a ca. 10-20 ns time scale, and Phe45 are broadened in all crystals, presumably due to µs motion. Our findings suggest that intramolecular influences are more important for ring flips than intermolecular (packing) effects., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)