Conformational Ensemble of Monomeric α-Synuclein in Aqueous and Crowded Environments as revealed by Markov State Model

140-residue intrinsically disordered protein -synuclein (S) is known to be susceptible to environmental cues/crowders and adopts conformations that are vastly variable in the extent of secondary structure and tertiary interactions. Depending upon the nature of these interactions, some of the conformations may be suitable for its physiological functions while some may be predisposed to aggregate with other partners into higher ordered species or to phase separate. However, the inherently heterogenous and dynamic nature of S has precluded a clear demarcation of its monomeric precursor between aggregation-prone and functionally relevant aggregation-resistant states. Here, we optimally characterise a set of metastable conformations of S by developing a comprehensive Markov state model (MSM) using cumulative 108 {micro}s-long all-atom MD simulation trajectories of monomeric S. Notably, the dimension of the most populated metastable (85%) state (Rg [~] 2.59 ({+/-}0.45) nm) corroborates PRENMR studies of S monomer and undergoes kinetic transition at 0.1-150 {micro}s time-scale with weakly populated (0.06%) random-coil like ensemble (Rg [~] 5.85 ({+/-}0.43) nm) and globular protein-like state (14%) (Rg [~] 1.95 ({+/-}0.08) nm). The inter-residue contact maps identify a set of mutually interconverting aggregation-prone {beta}-sheet networks in the NAC region and aggregation-resistant long-range interactions between N- and C-terminus or helical conformations. The presence of crowding agents compacts the MSM-derived metastable conformations in a non-monotonic fashion and skews the ensemble by either introducing new tertiary contacts or reinforcing the innate contacts to adjust to the excluded-volume effects of such environments. These observations of crucial monomeric states would serve as important steps towards rationalising routes that trigger S-associated pathologies. Significance statement-synuclein, a neuronal protein, is often associated with neurogenerative diseases due to its tendency to self-assemble into higher ordered aggregates. While the monomeric precursor of this protein is intrinsically disordered, it is also known to be susceptible to biological environmental cues and adopts a wide range of conformations that are either primed for aggregation or remain in auto-inhibitory states. However, the inherently heterogenous nature of the monomeric form has prevented a clear dissection of aggregation-prone and functionally relevant aggregation-resistant states. Here, we resolve this via an atomistic characterisation of an optimal set of crucial metastable monomeric conformations via statistical modelling of computer simulated data. The investigation also sheds light on crowding-induced modulation of the ensemble and eventual fibrillation pathways.