Biology subjects

Rauh, A. S.

Publications and source records attributed to Rauh, A. S..

3 recordsLinked to original sources

bioRxiv2026

On the Use of Double Mutant Cycles to Probe the Molecular Interactions in Biomolecular Condensates

Disordered proteins can form biomolecular condensates by demixing from their environment, enabling reversible compartmentalisation of cellular components in the form of membraneless organelles. Multivalent interactions are essential for this type of phase separation behaviour, and for disordered proteins, the potential for multivalent interactions is encoded in the sequence composition and patterning. Mutational studies have been instrumental in helping elucidate this sequence grammar by perturbing the amino acid sequence and quantifying the resulting changes in the driving force for phase separation. While such studies have provided a detailed and predictive understanding of the driving forces for phase separation, they strictly do not inform on the nature of the interactions that drive phase separation. Here, we propose using double mutant cycles to explore molecular interactions and their contributions to condensate properties more directly. We explore the applicability of double mutant cycles for different types of interactions in condensates formed by the low-complexity domain of hnRNPA1 using coarse-grained molecular dynamics simulations. We find that the interactions between arginine and tyrosine residues, as well as between aromatic residues, contribute mostly additively to the propensity for phase separation. However, for the interactions between charged residues, we find that--in an interplay with the net charge of the protein--there is a measurable non-additive contribution to the phase separation propensity. Based on our results, we envisage that double mutant cycles could provide additional insights into protein phase separation, thus expanding the understanding of the sequence grammar and the underlying molecular interactions.

biophysics↗

bioRxiv2025

A coarse-grained model for simulations of phosphorylated disordered proteins

Protein phosphorylation is a common and essential post-translational modification that affects biochemical properties and regulates biological activities. Phosphorylation is particularly common for intrinsically disordered proteins and can significantly modulate their function and potential to interact with binding partners. To understand the biophysical origins of how phosphorylation of disordered proteins influences their function, it is valuable to investigate how the modifications lead to changes in their conformational ensembles. Here, we have used a top-down data-driven approach to develop a coarse-grained molecular dynamics model compatible with the CALVADOS protein simulation model to study the effects of serine and threonine phosphorylation on the global structural properties of disordered proteins. We parameterise the model using experimental data on the effects of phosphorylation on global dimensions. By comparing with baseline models and simulations using the phosphomimetics aspartate and glutamate, we show that the effect of phosphorylation on the global dimensions of disordered proteins is mostly driven by the additional charge. We envisage that our model can be applied to study the effects of phosphorylation of disordered proteins at the proteome scale as well as to study the important roles of protein phosphorylation on phase separation.

biophysics↗

bioRxiv2025

A coarse-grained model for disordered proteins under crowded conditions

Macromolecular crowding may strongly affect the dynamics and function of proteins, with intrinsically disordered proteins being particularly sensitive to their crowded environment. To understand the influences of crowding on chain compaction and phase separation behaviour of disordered proteins, both experiments with synthetic crowders--like polyethylene glycol (PEG) and ficoll--and theoretical models and molecular simulation approaches have been applied. Here, we developed a residue-based coarse-grained model for PEG that is compatible with the protein CALVADOS model. To achieve this, we optimised model parameters by comparing simulations with experimental data on single-chain PEG and on PEG-induced compaction of disordered proteins. With our model we show how titrations of PEG can be used to quantify phase separation propensities of proteins that are not prone to phase separate strongly. We illustrate this for both variants of the low-complexity domain of hnRNPA1, and for wild-type and a redesigned variant of -Synuclein. Notably, we observe that the PEG crowding response changes between charge patterning variants of -Synuclein, which is not the case for the variants that vary the number the of aromatic residues in the low-complexity domain of hnRNPA1. We expect that our model will be useful for the interpretation of crowding experiments with disordered proteins, and we envisage it to be a starting point for in-silico explorations of proteins with weak propensities to phase separate.

biophysics↗