Dynamics, kinetics and assembly of model intrinsically disordered proteins from a polymer physics perspective – IDPXN

The IDPXN project has combined experimental and simulation studies of non-Fickian diffusion within the highly dense interior of β-casein assemblies which represent a model for super-crowded condensates of intrinsically disordered proteins. In a wide range around typical physiological protein concentrations, hydrodynamic interactions mediate protein-protein interactions already on the nanosecond time scale well before the onset of direct interactions. In terms of colloid physics, this hydrodynamic regime is denoted the regime of short-time diffusion, and even the short-time tracer diffusion of proteins in models of polydispersely crowded living cells has been found to agree with simple Fickian diffusion [M. Grimaldo et al., J.Phys.Chem.Lett. 10, 1709 (2019) and C. Beck et al., J.Phys.Chem. B 126, 7400 (2022)]. Yet unexplored has been the situation of extreme crowding that exceeds the average level of crowding in living cells but occurs locally in biomolecular condensates.

The IDPXN project has combined experiments using the most advanced x-ray and neutron scattering instrumentation including experiments using Megahertz x-ray photon correlation spectroscopy at the European Free Electron Laser and high-resolution neutron spectroscopy using novel fast pulse chopper instrumentation. These experiments, in combination accessing pico- to microsecond time scales on nanometer length scales, have helped to characterize β-casein and other reference proteins in aqueous solution. As a result of this endeavor, a model of super-crowded IDP condensates has emerged, represented by β-casein assemblies, showing that the tracer diffusion of the individual proteins within the condensates deviates from simple Fickian diffusion. The observed systematic deviation phenomenologically agrees with the so-called Singwi-Sjölander jump diffusion previously established for the diffusion in liquids and can be understood by dynamic heterogeneity and non-Gaussian terms. This model of dynamic heterogeneity and non-Gaussian contributions explains both the experimental and simulated data for the protein diffusion within the dense β-casein assemblies (see the figure below).

The findings from the project address fundamental aspects of the physics of diffusion in many-particle systems as well as its impact on complex fluids consisting of dense assemblies of flexible macromolecules which include biomolecular condensates. The findings will be of interest for experimental and theoretical physicists, physical chemists, biologists as well as for scientists investigating the dynamics of biomolecular condensates from the perspective of the medical sciences. The central scientific publications are in the process of peer-review at the closing of the project.

Supramolecular assemblies of intrinsically disordered proteins (IDPs), i.e. of proteins lacking a stable 3D-structure, are important in cellular signaling and spatial organization and can be involved in pathologies. Here, we wish to explore the dynamics and kinetics of such assembly processes of IDPs to understand the fundamental mechanisms behind IDP-related diseases. Using the model systems a, ß, and kappa-casein (all disordered), ß-lactoglobulin and apo-myoglobin (both ordered), we intend to establish their respective phase diagrams in solution, to determine the parameter space leading to the formation of supramolecular IDP assemblies, and to quantify the influence of protein disorder on these assembly pathways, as well as to compare chemically and thermally denatured proteins. This project systematically combines advanced x-ray and neutron scattering methods, notably accessing dynamics on the molecular level during kinetics, as well as theory.