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Biomolecules are in constant motion. To understand how they function, and why malfunctions can cause disease, it is necessary to describe their three-dimensional structures in terms of dynamic conformational ensembles. Here, we demonstrate how nuclear magnetic resonance (NMR) spectroscopy provides an essential, dynamic view of structural biology that captures biomolecular motions at atomic resolution. We focus on examples that emphasize the diversity of biomolecules and biochemical applications that are amenable to NMR, such as elucidating functional dynamics in large molecular machines, characterizing transient conformations implicated in the onset of disease, and obtaining atomic-level descriptions of intrinsically disordered regions that make weak interactions involved in liquid-liquid phase separation. Finally, we discuss the pivotal role that NMR has played in driving forward our understanding of the biomolecular dynamics-function paradigm., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

As structural biology trends towards larger and more complex biomolecular targets, a detailed understanding of their interactions and underlying structures and dynamics is required. The development of methyl-TROSY has enabled NMR spectroscopy to provide atomic-resolution insight into the mechanisms of large molecular assemblies in solution. However, the applicability of methyl-TROSY has been hindered by the laborious and time-consuming resonance assignment process, typically performed with domain fragmentation, site-directed mutagenesis, and analysis of NOE data in the context of a crystal structure. In response, several structure-based automatic methyl assignment strategies have been developed over the past decade. Here, we present a comprehensive analysis of all available methods and compare their input data requirements, algorithmic strategies, and reported performance. In general, the methods fall into two categories: those that primarily rely on inter-methyl NOEs, and those that utilize methyl PRE- and PCS-based restraints. We discuss their advantages and limitations, and highlight the potential benefits from standardizing and combining different methods., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Pulsed-field gradient NMR spectroscopy is widely used to measure the translational diffusion and hydrodynamic radius (R h ) of biomolecules in solution. For unfolded proteins, the R h provides a sensitive reporter on the ensemble-averaged conformation and the extent of polypeptide chain expansion as a function of added denaturant. Hydrostatic pressure is a convenient and reversible alternative to chemical denaturants for the study of protein folding, and enables NMR measurements to be performed on a single sample. While the impact of pressure on the viscosity of water is well known, and our water diffusivity measurements agree closely with theoretical expectations, we find that elevated pressures increase the R h of dioxane and other small molecules by amounts that correlate with their hydrophobicity, with parallel increases in rotational friction indicated by 13 C longitudinal relaxation times. These data point to a tighter coupling with water for hydrophobic surfaces at elevated pressures. Translational diffusion measurement of the unfolded state of a pressure-sensitized ubiquitin mutant (VA2-ubiquitin) as a function of hydrostatic pressure or urea concentration shows that R h values of both the folded and the unfolded states remain nearly invariant. At ca 23 Å, the R h of the fully pressure-denatured state is essentially indistinguishable from the urea-denatured state, and close to the value expected for an idealized random coil of 76 residues. The intrinsically disordered protein (IDP) α-synuclein shows slight compaction at pressures above 2 kbar. Diffusion of unfolded ubiquitin and α-synuclein is significantly impacted by sample concentration, indicating that quantitative measurements need to be carried out under dilute conditions., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Proteins interconvert between multiple conformations, including sparsely populated and transiently formed states that are difficult to characterize in structural detail using standard biophysical methods. In some cases, changes to the dynamical equilibria between conformations can lead to pathological protein aggregation and to the disruption of cellular homeostasis. The detection and characterization of lowly populated conformers is therefore crucial for understanding the basis of protein misfolding. NMR spectroscopy is exquisitely sensitive to the conformational dynamics of biomolecules and can be used to study sparsely populated states at the atomic level. Here, we review recent progress toward understanding the roles of sparsely populated, otherwise 'invisible' states present in protein folding and misfolding, where NMR has provided unique insight into folding intermediates, transiently misfolded states, and soluble oligomers that precede amyloid fibril formation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Isotopically labeled methyl groups provide NMR probes in large, otherwise deuterated proteins. However, the resonance assignment constitutes a bottleneck for broader applicability of methyl-based NMR. Here, we present the automated MethylFLYA method for the assignment of methyl groups that is based on methyl-methyl nuclear Overhauser effect spectroscopy (NOESY) peak lists. MethylFLYA is applied to five proteins (28-358 kDa) comprising a total of 708 isotope-labeled methyl groups, of which 612 contribute NOESY cross peaks. MethylFLYA confidently assigns 488 methyl groups, i.e. 80% of those with NOESY data. Of these, 459 agree with the reference, 6 were different, and 23 were without reference assignment. MethylFLYA assigns significantly more methyl groups than alternative algorithms, has an average error rate of 1%, modest runtimes of 0.4-1.2 h, and can handle arbitrary isotope labeling patterns and data from other types of NMR spectra.

Pressure-jump hardware permits direct observation of protein NMR spectra during a cyclically repeated protein folding process. For a two-state folding protein, the change in resonance frequency will occur nearly instantaneously when the protein clears the transition state barrier, resulting in a monoexponential change of the ensemble-averaged chemical shift. However, protein folding pathways can be more complex and contain metastable intermediates. With a pseudo-3D NMR experiment that utilizes stroboscopic observation, we measure the ensemble-averaged chemical shifts, including those of exchange-broadened intermediates, during the folding process. Such measurements for a pressure-sensitized mutant of ubiquitin show an on-pathway kinetic intermediate whose 15 N chemical shifts differ most from the natively folded protein for strands β5, its preceding turn, and the two strands that pair with β5 in the native structure.

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. 15 N and 13 C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, 15 N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

A method is introduced that permits direct observation of the rates at which backbone amide hydrogens become protected from solvent exchange after rapidly dropping the hydrostatic pressure inside the NMR sample cell from denaturing (2.5 kbar) to native (1 bar) conditions. The method is demonstrated for a pressure-sensitized ubiquitin variant that contains two Val to Ala mutations. Increased protection against hydrogen exchange with solvent is monitored as a function of time during the folding process. Results for 53 backbone amides show narrow clustering with protection occurring with a time constant of ca. 85 ms, but slower protection is observed around a reverse turn near the C-terminus of the protein. Remarkably, the native NMR spectrum returns with this slower time constant of ca. 150 ms, indicating that the almost fully folded protein retains molten globule characteristics with severe NMR line broadening until the final hydrogen bonds are formed. Prior to crossing the transition state barrier, hydrogen exchange protection factors are close to unity, but with slightly elevated values in the β 1 -β 2 hairpin, previously shown to be already lowly populated in the urea-denatured state.

Methyl groups are powerful probes for the analysis of structure, dynamics and function of supramolecular assemblies, using both solution- and solid-state NMR. Widespread application of the methodology has been limited due to the challenges associated with assigning spectral resonances to specific locations within a biomolecule. Here, we present Methyl Assignment by Graph Matching (MAGMA), for the automatic assignment of methyl resonances. A graph matching protocol examines all possibilities for each resonance in order to determine an exact assignment that includes a complete description of any ambiguity. MAGMA gives 100% accuracy in confident assignments when tested against both synthetic data, and 9 cross-validated examples using both solution- and solid-state NMR data. We show that this remarkable accuracy enables a user to distinguish between alternative protein structures. In a drug discovery application on HSP90, we show the method can rapidly and efficiently distinguish between possible ligand binding modes. By providing an exact and robust solution to methyl resonance assignment, MAGMA can facilitate significantly accelerated studies of supramolecular machines using methyl-based NMR spectroscopy.