Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes, play a major role in cell signaling, and are associated with human diseases. To understand IDP function it is critical to determine their configurational ensemble, i.e., the collection of 3-dimensional structures they adopt, and this remains an immense challenge in structural biology. Attempts to determine this ensemble computationally have been hitherto hampered by the necessity of reweighting molecular dynamics (MD) results or biasing simulation in order to match ensemble-averaged experimental observables, operations that reduce the precision of the generated model because different structural ensembles may yield the same experimental observable. Here, by employing enhanced sampling MD we reproduce the experimental small-angle neutron and X-ray scattering profiles and the NMR chemical shifts of the disordered N terminal (SH4UD) of c-Src kinase without reweighting or constraining the simulations. The unbiased simulation results reveal a weakly funneled and rugged free energy landscape of SH4UD, which gives rise to a heterogeneous ensemble of structures that cannot be described by simple polymer theory. SH4UD adopts transient helices, which are found away from known phosphorylation sites and could play a key role in the stabilization of structural regions necessary for phosphorylation. Our findings indicate that adequately sampled molecular simulations can be performed to provide accurate physical models of flexible biosystems, thus rationalizing their biological function.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Light activation of the visual G-protein-coupled receptor rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane. Enhancement of protein dynamics upon stimulation of the GPCR yields activation of the cognate G-protein (transducin) that initiates biological signaling. Although X-ray crystallographic analysis reveals static structures, changes in protein dynamics are the key to understanding the activation mechanism. Here we show how the integral membrane protein mobility is regulated by the retinal cofactor of the visual GPCR rhodopsin using both elastic and quasi-elastic neutron scattering. Our quasi-elastic neutron scattering (QENS) experiments revealed a logarithmic-like relaxation of the hydrogen-atom dynamics in the Rhodopsin family A GPCRs, as only observed for globular proteins previously. Application of mode-coupling theory (MCT) as originally developed for glass-forming liquids to our QENS analysis reveals the picosecond–nanosecond dynamics in the β-relaxation region crucial to protein function. Our novel powdered GPCR preparation method together with the QENS technique allowed us to uncover subtle changes in protein dynamics regulated by the retinal cofactor of rhodopsin. For the ligand-free opsin apoprotein versus the dark-state rhodopsin, removal of the retinal cofactor increases the relaxation time in the β-relaxation regime (ps–ns), evincing greater protein flexibility. Because opsin is structurally similar to active metarhodopsin-II, which catalytically activates transducin, the cofactor plays a pivotal role in regulating the protein dynamics required for GPCR function.

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD (HREMD) led to unbiased and accurate ensembles, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of varying sequence properties using two recently optimized force fields, indicating the general applicability of HREMD for IDPs. We further demonstrate that, unlike HREMD, standard MD can reproduce experimental NMR chemical shifts, but not small-angle scattering data, suggesting chemical shifts are insufficient for testing the validity of IDP ensembles. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues). The results suggest that the major hurdle of generating an accurate unbiased ensemble for IDPs has now been largely overcome.

Inorganic pyrophosphatase (IPPase) from Thermococcus thioreducens is a large oligomeric protein derived from a hyperthermophilic microorganism that is found near hydrothermal vents deep under the sea, where the pressure is up to 100 MPa (1 kbar). It has attracted great interest in biophysical research because of its high activity under extreme conditions in the seabed. In this study, we use the quasielastic neutron scattering (QENS) technique to investigate the effects of pressure on the conformational flexibility and relaxation dynamics of IPPase over a wide temperature range. The β-relaxation dynamics of proteins was studied in the time ranges from 2 to 25 ps, and from 100 ps to 2 ns, using two spectrometers. Our results indicate that, under a pressure of 100 MPa, close to that of the native environment deep under the sea, IPPase displays much faster relaxation dynamics than a mesophilic model protein, hen egg white lysozyme (HEWL), at all measured temperatures, opposite to what we observed previously under ambient pressure. This contradictory observation provides evidence that the protein energy landscape is distorted by high pressure, which is significantly different for hyperthermophilic (IPPase) and mesophilic (HEWL) proteins. We further derive from our observations a schematic denaturation phase diagram together with energy landscapes for the two very different proteins, which can be used as a general picture to understand the dynamical properties of thermophilic proteins under pressure.protein dynamics | energy landscape | denaturation phase diagram | quasielastic neutron scattering | mode coupling theory T he biological functions of proteins, such as enzyme catalysis, are often understood from their crystallographic structures (1). On the other hand, it is crucial to take into account dynamic behavior to fully comprehend these functions (2, 3). In vivo, proteins are in constant motion among different conformations (3-5). The thermal energy, which is of the order of k B T per atom, where k B is the Boltzmann constant and T is the absolute temperature, triggers biomolecules to sample different conformations around the average structure. These conformations are also known as conformational substates (CSs) (5). Fluctuations among these CSs play an important role in protein function (3,5). These lead to the concept of a multidimensional potential energy landscape (EL) that specifies a complete description of CSs in proteins (6-9). The existence of an EL was proposed by H. Frauenfelder and others in the 1970s and has been validated both by computations and by experiments (6-12).Proteins show various dynamic phenomena over a wide range of timescales, from picoseconds to milliseconds (13). A fast dynamic process, on a timescale of a picosecond to 10 ns, also known as β-relaxation, occurs due to small amplitude fluctuations in atoms/ molecules, such as loop motions and side-chain rotations (14). The energy barrier or activation energy (E A ) between different CSs for this process is smaller than k B T (15). On t...