Abstract
Intrinsically disordered proteins (IDPs) are proteins that, in comparison with globular/structured proteins, lack a distinct tertiary structure. Here, we use the model IDP, Histatin 5, for studying its dynamical properties under self-crowding conditions with quasi-elastic neutron scattering in combination with full atomistic molecular dynamics (MD) simulations. The aim is to determine the effects of crowding on the center-of-mass diffusion as well as the internal diffusive behavior. The diffusion was found to decrease significantly, which we hypothesize can be attributed to some degree of aggregation at higher protein concentrations, (≥100 mg/mL), as indicated by recent small-angle X-ray scattering studies. Temperature effects are also considered and found to, largely, follow Stokes–Einstein behavior. Simple geometric considerations fail to accurately predict the rates of diffusion, while simulations show semiquantitative agreement with experiments, dependent on assumptions of the ratio between translational and rotational diffusion. A scaling law that previously was found to successfully describe the behavior of globular proteins was found to be inadequate for the IDP, Histatin 5. Analysis of the MD simulations show that the width of the distribution with respect to diffusion is not a simplistic mirroring of the distribution of radius of gyration, hence, displaying the particular features of IDPs that need to be accounted for.
Highlights
In contrast to globular proteins, intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure, instead they adopt an ensemble of conformers in solution.[1]
It is expected that protein− protein interactions, as excluded volume effects and electrostatic interactions, impact the conformational ensemble,[8−10] and restrict the ability of IDPs to diffuse throughout the crowded intracellular milieu.[11]
The data point for 50 mg/mL and 310 K is missing in the data set since we, for the IDP of the size used in this study, reached the limitations of technique at IN16B; i.e., the combined high speed-diffusion and comparably low protein concentrations did not provide feasible data
Summary
In contrast to globular proteins, intrinsically disordered proteins (IDPs) lack a well-defined three-dimensional structure, instead they adopt an ensemble of conformers in solution.[1]. The precise nature of the conformational ensembles adopted by IDPs depend on a variety of conditions including, for example, temperature, ionic strength, and presence of binding partners.[4,5] One condition, often neglected by experimental studies, is the effect of crowding on the dynamical properties of IDPs, and how these effects relate to the protein function. Determining the dynamical properties under crowded conditions is pertinent due to the high intracellular concentration of macromolecules, which can reach up to 400 mg/mL.[6,7] At these concentrations, it is expected that protein− protein interactions, as excluded volume effects and electrostatic interactions, impact the conformational ensemble,[8−10] and restrict the ability of IDPs to diffuse throughout the crowded intracellular milieu.[11]
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