Protein function depends critically on intrinsic internal dynamics, which is manifested in distinct ways, such as loop motions that regulate protein recognition and catalysis. Under physiological conditions, dynamic processes occur on a wide range of time scales from subpicoseconds to seconds. Commonly used NMR spin relaxation in solution provides valuable information on very fast and slow motions but is insensitive to the intermediate nanosecond to microsecond range that exceeds the protein tumbling correlation time. Presently, very little is known about the nature and functional role of these motions. It is demonstrated here how transverse spin relaxation becomes exquisitely sensitive to these motions at atomic resolution when studying proteins in the presence of nanoparticles. Application of this novel cross-disciplinary approach reveals large-scale dynamics of loops involved in functionally critical protein-protein interactions and protein-calcium ion recognition that were previously unobservable.

Molecular dynamics (MD) simulations provide a unique atomic-level description of the structure and dynamics of proteins, which is essential for the mechanistic understanding of protein interactions and function in living organisms. However, traditional MD force fields that are optimized for folded proteins often generate overly compact structures and incorrect characteristics of intrinsically disordered proteins (IDPs) and protein regions (IDRs), thereby limiting the quantitative insights that can be gained from MD simulations. We introduce the residue-specific protein force field, ff99SBnmr2, which is derived from ff99SBnmr1 by balancing the backbone dihedral angle potentials in a residuespecific manner to quantitatively reproduce dihedral angle distributions from an experimental coil library. The new force field substantially improves the backbone conformational ensembles of disordered proteins, protein regions, and peptides while keeping well-defined protein structures stable and accurate. This balanced new force field should enable a myriad of applications that require quantitative descriptions of IDPs, IDRs, loop dynamics, and folding/unfolding equilibria in the presence and absence of interaction partners.

The prevalence of intrinsically disordered proteins (IDPs) and protein regions in structural biology has prompted the recent development of molecular dynamics (MD) force fields for the more realistic representations of such systems. Using experimental nuclear magnetic resonance backbone scalar 3 J-coupling constants of the intrinsically disordered proteins αsynuclein and amyloid-β in their native aqueous environment as a metric, we compare the performance of four recent MD force fields, namely, AMBER ff14SB, CHARMM C36m, AMBER ff99SB-disp, and AMBER ff99SBnmr2, by partitioning the polypeptides into an overlapping series of heptapeptides for which a cumulative total of 276 μs MD simulations were performed. The results show substantial differences between the different force fields at the individual residue level. Except for ff99SBnmr2, the force fields systematically underestimate the scalar 3 J(H N ,Hα)-couplings due to an underrepresentation of β-conformations and an overrepresentation of either αor PP II conformations. The study demonstrates that the incorporation of coil library information in modern MD force fields, as shown here for ff99SBnmr2, provides substantially improved performance and more realistic sampling of the local backbone dihedral angles of IDPs as reflected by the good accuracy of the computed scalar 3 J(H N ,Hα)-couplings with less than 0.5 Hz error. Such force fields will enable a better understanding of how structural dynamics and thermodynamics influence the IDP function. Although the methodology based on heptapeptides used here does not allow the assessment of potential intramolecular long-range interactions, its computational affordability permits well-converged simulations that can be easily parallelized. This should make the quantitative validation of intrinsic disorder observed in MD simulations of polypeptides with experimental scalar J-couplings widely applicable.

The power of structural information for informing biological mechanisms is clear for stable folded macromolecules, but similar structure–function insight is more difficult to obtain for highly dynamic systems such as intrinsically disordered proteins (IDPs) which must be described as structural ensembles. Here, we present IDPConformerGenerator, a flexible, modular open-source software platform for generating large and diverse ensembles of disordered protein states that builds conformers that obey geometric, steric, and other physical restraints on the input sequence. IDPConformerGenerator samples backbone phi (φ), psi (ψ), and omega (ω) torsion angles of relevant sequence fragments from loops and secondary structure elements extracted from folded protein structures in the RCSB Protein Data Bank and builds side chains from robust Monte Carlo algorithms using expanded rotamer libraries. IDPConformerGenerator has many user-defined options enabling variable fractional sampling of secondary structures, supports Bayesian models for assessing the agreement of IDP ensembles for consistency with experimental data, and introduces a machine learning approach to transform between internal and Cartesian coordinates with reduced error. IDPConformerGenerator will facilitate the characterization of disordered proteins to ultimately provide structural insights into these states that have key biological functions.

The quantitative and comprehensive description of the internal dynamics of proteins is critical for understanding their function. Nanoparticle-assisted 15 N NMR spin relaxation spectroscopy is a new method for the observation of picosecond to microsecond dynamics of proteins when transiently interacting with the surface of the nanoparticles (NPs). The method is applied here to the protein ubiquitin in the presence of anionic and cationic silica NPs (SNPs) of different sizes. The backbone dynamics profiles are reproducible and strikingly similar to each other, indicating that specific protein-SNP interactions are unimportant. The dynamics profiles closely match the sub-nanosecond dynamics S 2 values observed by model-free analysis of standard 15 N relaxation of ubiquitin in free solution, indicating that the bulk of the ubiquitin backbone dynamics in solution is confined to sub-nanosecond timescales and, hence, it is dynamically more restrained than previous NMR studies have suggested.