We report an analysis of high-resolution quasielastic neutron scattering spectra from Myelin Basic Protein (MBP) in solution, comparing the spectra at three different temperatures (283, 303, and ...323 K) for a pure D
O buffer and a mixture of D
O buffer with 30% of deuterated trifluoroethanol (TFE). Accompanying experiments with dynamic light scattering and Circular Dichroism (CD) spectroscopy have been performed to obtain, respectively, the global diffusion constant and the secondary structure content of the molecule for both buffers as a function of temperature. Modeling the decay of the neutron intermediate scattering function by the Mittag-Leffler relaxation function, ϕ(t) = E
(-(t/τ)
) (0 < α < 1), we find that trifluoroethanol slows down the relaxation dynamics of the protein at 283 K and leads to a broader relaxation rate spectrum. This effect vanishes with increasing temperature, and at 323 K, its relaxation dynamics is identical in both solvents. These results are coherent with the data from dynamic light scattering, which show that the hydrodynamic radius of MBP in TFE-enriched solutions does not depend on temperature and is only slightly smaller compared to the pure D
O buffer, except for 283 K, where it is much reduced. In accordance with these observations, the CD spectra reveal that TFE induces essentially a partial transition from β-strands to α-helices, but only a weak increase in the total secondary structure content, leaving about 50% of the protein unfolded. The results show that MBP is for all temperatures and in both buffers an intrinsically disordered protein and that TFE essentially induces a reduction in its hydrodynamic radius and its relaxation dynamics at low temperatures.
Intrinsically disordered proteins lack a well-defined folded structure and contain a high degree of structural freedom and conformational flexibility, which is expected to enhance binding to their ...physiological targets. In solution and in the lipid-free state, myelin basic protein belongs to that class of proteins. Using small-angle scattering, the protein was found to be structurally disordered similar to Gaussian chains. The combination of structural and hydrodynamic information revealed an intermediary compactness of the protein between globular proteins and random coil polymers. Modeling by a coarse-grained structural ensemble gave indications for a compact core with flexible ends. Neutron spin–echo spectroscopy measurements revealed a large contribution of internal dynamics to the overall diffusion. The experimental results showed a high flexibility of the structural ensemble. Displacement patterns along the first two normal modes demonstrated that collective stretching and bending motions dominate the internal modes. The observed dynamics represent nanosecond conformational fluctuations within the reconstructed coarse-grained structural ensemble, allowing the exploration of a large configurational space. In an alternative approach, we investigated if models from polymer theory, recently used for the interpretation of fluorescence spectroscopy experiments on disordered proteins, are suitable for the interpretation of the observed motions. Within the framework of the Zimm model with internal friction (ZIF), a large offset of 81.6 ns is needed as an addition to all relaxation times due to intrachain friction sources. The ZIF model, however, shows small but systematic deviations from the measured data. The large value of the internal friction leads to the breakdown of the Zimm model.
A general property of disordered proteins is their structural expansion that results in a high molecular flexibility. The structure and dynamics of bovine serum albumin (BSA) denatured by guanidinium ...hydrochloride (GndCl) were investigated using small-angle neutron scattering (SANS) and neutron spin–echo spectroscopy (NSE). SANS experiments demonstrated the relevance of intrachain interactions for structural expansion. Using NSE experiments, we observed a high internal flexibility of denatured BSA in addition to center-of-mass diffusion detected by dynamic light scattering. Internal motions measured by NSE were described using concepts based on polymer theory. The contribution of residue-solvent friction was accounted for using the Zimm model including internal friction (ZIF). Disulfide bonds forming loops of amino acids of the peptide backbone have a major impact on internal dynamics that can be interpreted with a reduced set of Zimm modes.
Small-angle X-ray and small-angle neutron scattering (SAXS/SANS) provide unique structural information on biomolecules and their complexes in solution. SANS may provide multiple independent data sets ...by means of contrast variation experiments, that is, by measuring at different D2O concentrations and different perdeuteration conditions of the biomolecular complex. However, even the combined data from multiple SAXS/SANS sets is by far insufficient to define all degrees of freedom of a complex, leading to a significant risk of overfitting when refining biomolecular structures against SAXS/SANS data. Hence, to control against overfitting, the low-information SAXS/SANS data must be complemented by accurate physical models, and, if possible, refined models should be cross-validated against independent data not used during the refinement. We present a method for refining atomic biomolecular structures against multiple sets of SAXS and SANS data using all-atom molecular dynamics simulations. Using the protein citrate synthase and the protein/RNA complex Sxl–Unr–msl2 mRNA as test cases, we demonstrate how multiple SAXS and SANS sets may be used for refinement and cross-validation, thereby excluding overfitting during refinement. For the Sxl–Unr–msl2 complex, we find that perdeuteration of the Unr domain leads to a unique, slightly compacted conformation, whereas other perdeuteration conditions lead to similar solution conformations compared to the nondeuterated state. In line with our previous method for predicting SAXS curves, SANS curves were predicted with explicit-solvent calculations, taking atomic models for both the hydration layer and the excluded solvent into account, thereby avoiding the use of solvent-related fitting parameters and solvent-reduced neutron scattering lengths. We expect the method to be useful for deriving and validating solution structures of biomolecules and soft-matter complexes, and for critically assessing whether multiple SAXS and SANS sets are mutually compatible.
The bacterial flagellar filament is a very large macromolecular assembly of a single protein, flagellin. Various supercoiled states of the filament exist, which are formed by two structurally ...different conformations of flagellin in different ratios. We investigated the correlation between supercoiling of the protofilaments and molecular dynamics in the flagellar filament using quasielastic and elastic incoherent neutron scattering on the picosecond and nanosecond timescales. Thermal fluctuations in the straight L- and R-type filaments were measured and compared to the resting state of the wild-type filament. Amplitudes of motion on the picosecond timescale were found to be similar in the different conformational states. Mean-square displacements and protein resilience on the 0.1 ns timescale demonstrate that the L-type state is more flexible and less resilient than the R-type, whereas the wild-type state lies in between. Our results provide strong support that supercoiling of the protofilaments in the flagellar filament is determined by the strength of molecular forces in and between the flagellin subunits.
A characteristic property of unfolded and disordered proteins is their high molecular flexibility, which enables the exploration of a large conformational space. We present neutron scattering ...experiments on the dynamics of denatured and native folded bovine serum albumin (BSA) in solution. Global protein diffusion and internal macromolecular dynamics were measured using quasielastic neutron time-of-flight and backscattering spectroscopy on the picosecond to nanosecond time- and Ångstrom length-scale. Internal protein dynamics were analysed in a first approach using stretched exponential functions. In denatured BSA predominantly slow heterogeneous dynamics dominates the observed macromolecular motions. Reduction of disulphide bridges in denatured BSA does not significantly alter the visible motions. In native folded BSA fast homogeneous dynamics and slow heterogeneous dynamics were observed. In an alternative data analysis approach, internal protein dynamics was interpreted using the analytical model of the overdamped Brownian oscillator, which allowed us to extract mean square displacements of protein internal dynamics and the fraction of hydrogen atoms participating in the observed motions. Our results demonstrate that denaturation modifies the physical nature of internal protein dynamics significantly as compared to the native folded structure.
We used neutron-scattering experiments to probe the conformational dynamics of the light, oxygen, voltage (LOV) photoreceptor PpSB1-LOV from Pseudomonas putida in both the dark and light states. ...Global protein diffusion and internal macromolecular dynamics were measured using incoherent neutron time-of-flight and backscattering spectroscopy on the picosecond to nanosecond timescales. Global protein diffusion of PpSB1-LOV is not influenced by photoactivation. Observation-time-dependent global diffusion coefficients were found, which converge on the nanosecond timescale toward diffusion coefficients determined by dynamic light scattering. Mean-square displacements of localized internal motions and effective force constants, <k′>, describing the resilience of the proteins were determined on the respective timescales. Photoactivation significantly modifies the flexibility and the resilience of PpSB1-LOV. On the fast, picosecond timescale, small changes in the mean-square displacement and <k′> are observed, which are enhanced on the slower, nanosecond timescale. Photoactivation results in a slightly larger resilience of the photoreceptor on the fast, picosecond timescale, whereas in the nanosecond range, a significantly less resilient structure of the light-state protein is observed. For a residue-resolved interpretation of the experimental neutron-scattering data, we analyzed molecular dynamics simulations of the PpSB1-LOV X-ray structure. Based on these data, it is tempting to speculate that light-induced changes in the protein result in altered side-chain mobility mostly for residues on the protruding Jα helix and on the LOV-LOV dimer interface. Our results provide strong experimental evidence that side-chain dynamics play a crucial role in photoactivation and signaling of PpSB1-LOV via modulation of conformational entropy.
Molecular dynamics plays an important role for the biological function of proteins. For protein ligand interactions, changes of conformational entropy of protein and hydration layer are relevant for ...the binding process. Quasielastic neutron scattering (QENS) was used to investigate differences in protein dynamics and conformational entropy of ligand-bound and ligand-free streptavidin. Protein dynamics were probed both on the fast picosecond time scale using neutron time-of-flight spectroscopy and on the slower nanosecond time scale using high-resolution neutron backscattering spectroscopy. We found the internal equilibrium motions of streptavidin and the corresponding mean square displacements (MSDs) to be greatly reduced upon biotin binding. On the basis of the observed MSDs, we calculated the difference of conformational entropy ΔS conf of the protein component between ligand-bound and ligand-free streptavidin. The rather large negative ΔS conf value (−2 kJ mol–1 K–1 on the nanosecond time scale) obtained for the streptavidin tetramer seems to be counterintuitive, given the exceptionally high affinity of streptavidin–biotin binding. Literature data on the total entropy change ΔS observed upon biotin binding to streptavidin, which includes contributions from both the protein and the hydration water, suggest partial compensation of the unfavorable ΔS conf by a large positive entropy gain of the surrounding hydration layer and water molecules that are displaced during ligand binding.
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