Condensates formed by oppositely charged intrinsically disordered proteins provide model systems for understanding how transient electrostatic interactions govern structure and dynamics in biomolecular assemblies. Here we investigate a nearly charge-neutral condensate composed of 50 Prothymosin alpha (ProTalpha) and 40 Histone H1 molecules using a single-bead-per-residue coarse-grained model combining the HPS hydropathy model for disordered regions with a Go model for the globular domain of Histone H1 under NPT conditions at pressures from 2 to 12 bar. We find that chain dimensions, including the radius of gyration (Rg), end-to-end distance (Ree), and their ratio R, are insensitive to pressure, indicating that chain conformations remain largely unchanged over the pressure range studied. Histone H1 exhibits systematically larger values of R than ProTalpha because of its globular-core plus disordered-tail architecture. Translational diffusion coefficients decrease monotonically with pressure, from approximately 0.22 to 0.06 nm^2/ns, with substantial chain-to-chain heterogeneity comparable to the mean diffusion coefficient. Chain relaxation follows a stretched exponential with beta less than 1 that decreases with pressure. ProTalpha relaxation times of approximately 12 to 40 ns obey Rouse scaling, whereas Histone H1 deviates because of the internal constraint imposed by its globular domain. ProTalpha-Histone H1 contact lifetimes of approximately 0.43 to 0.56 ns are much shorter than the Rouse relaxation time, placing the system firmly in the fast-exchange regime where transient electrostatic contacts renormalize chain friction rather than acting as permanent cross-links, consistent with the moderate stretching exponent beta of approximately 0.55 to 0.70 observed across all pressures.
This work investigates how protein folding landscapes are altered inside condensates, using the protein α-helix as a model folded domain and develops a chemically specific, residue-resolution model for quantification of α-helical folding and applies it to characterize diverse helices within condensates of varying physicochemical properties.
Nathaniel Hess, Jerelle A. Joseph· Journal of the American Chem...· 0 citations
Physical remodeling of chromatin by non-histone architectural proteins of the High Mobility Group B (HMGB) family is central to eukaryotic transcriptional regulation. Nhp6A, the prototypical single-HMG-box protein from yeast, harbors both ordered and disordered regions enabling it to bind and bend DNA without sequence specificity. Here, we integrate ensemble experiments, single-molecule FRET, statistical mechanical modeling and atomistic simulations to dissect the structural and functional consequences of context-dependent phosphorylation in the ordered domain and its interplay with the intrinsically disordered region in Nhp6A. We find that Nhp6A occupies a narrow thermodynamic window, with a melting temperature close to the growth temperature of its host organism and high unfolding cooperativity, a feature conserved across the HMG-box family. Phosphorylation extents – mimicked by multisite phosphomimetic substitutions at residue positions conserved across fungal taxa – smoothly tuning the conformational equilibria between at least two different substates in the native ensemble, apart from the unfolded state. This intrinsic plasticity enables close packing of Nhp6A on DNA through two degenerate binding modes, accompanied by two distinct DNA bending geometries. DNA rescues a strongly destabilized mutant, T63D, through favorable intermolecular interactions, thus effectively acting as a chaperone driving folding. Our findings thus reveal a conserved sequence-ensemble-dynamics code in Nhp6A wherein not just stability, but also phosphorylation-induced conformational switching, disordered tail dynamics, and DNA binding-bending closely coordinate chromatin accessibility. The combination of marginal stability, large cooperativity and electrostatic frustration emerges as a design principle to encode charge sensitivity into proteins, and may represent a general strategy for multisite post-translational regulation.
Shilpi Laha, H. Madhan, Yuji Itoh et al.· bioRxiv· 0 citations
Molecular recognition by intrinsically disordered regions (IDRs) is widely thought to involve coupled folding and binding, yet the sequence features that regulate this transition remain underexplored. Here we show that helix 8 (H8), a disordered C-terminal segment of the SCF ubiquitin ligase adaptor Skp1, is intrinsically prevented from forming a stable helix by its own sequence grammar. Using an integrative approach to dissect its conformational dynamics, we find that H8 frequently nucleates helical structure but rarely propagates into a fully formed stable helix, populating instead a shallow metastable basin of helical intermediates. Contrary to conventional models of helix-coil exchange, where nucleation is rate limiting, helix initiation in H8 is readily accessible, while propagation is selectively suppressed by a glutamate-rich acidic patch. This acidic segment acts as a charge-sensitive conformational rheostat that limits helix extension and maintains H8 in a predominantly disordered state. As a result, H8 transiently samples partially helical conformations on the microsecond timescale without committing to a stable fold. We propose that this propagation-limited mechanism preserves conformational flexibility while maintaining recognition competence across a structurally diverse family of F-box binding partners. More broadly, our findings suggest that charged- hydrophobic-charged sequence patterning can encode conditional, context-dependent structure as a general organisational principle in intrinsically disordered proteomes.
D. Mitra, Simran Tolani, Amrita Bhattacharya et al.· bioRxiv· 0 citations
G3BP1 is a central scaffold of stress granules (SGs). Upon cellular stress, G3BP1 forms complex coacervates with translationally repressed mRNAs and recruits multiple RNA-binding proteins to form reversible biomolecular condensates. Persistent SGs are linked to age-dependent dynamical arrest and impaired disassembly. Here, we employ active and passive nanoscale rheology with optical tweezers to show that G3BP1 condensates evolve from being dominantly viscous fluids to dynamically arrested network glasses characterized by nanoscale caging and elastic memory. Integrating atomistic and coarse-grained simulations with experiments, we find that electrostatic interactions between the oppositely charged intrinsically disordered regions drive condensate ageing. RNA modulates these interactions in a length-and structure-dependent manner, delaying dynamic arrest, whereas Caprin-1 binding to the NTF2L domain has little effect. Together, these findings reveal how competing inter-IDR and IDR–RNA interactions govern condensate ageing and material-state transitions. The findings have broader implications for the regulation of SG dynamics in cells.