The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0–6.3 nm) than the MAD within typical helices (4.3–5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

Intrinsically disordered protein α-synuclein (αS) is implicated in Parkinson’s disease due to its aberrant aggregation propensity. In a bid to identify the traits of its aggregation, here we computationally simulate the multi-chain association process of αS in aqueous as well as under diverse environmental perturbations. In particular, the aggregation of αS in aqueous and varied environmental condition led to marked concentration differences within protein aggregates, resembling liquid-liquid phase separation (LLPS). Both saline and crowded settings enhanced the LLPS propensity. However, the surface tension of αS droplet responds differently to crowders (entropy-driven) and salt (enthalpy-driven). Conformational analysis reveals that the IDP chains would adopt extended conformations within aggregates and would maintain mutually perpendicular orientations to minimize inter-chain electrostatic repulsions. The droplet stability is found to stem from a diminished intra-chain interactions in the C-terminal regions of αS, fostering inter-chain residue-residue interactions. Intriguingly, a graph theory analysis identifies small-world-like networks within droplets across environmental conditions, suggesting the prevalence of a consensus interaction patterns among the chains. Together these findings suggest a delicate balance between molecular grammar and environment-dependent nuanced aggregation behavior of αS.