**1. Introduction**

Intrinsically disordered proteins (IDPs) play crucial regulatory roles in biological systems and lack a specific tertiary structure under physiological conditions [1–4]. Molecular characterization of IDPs requires description of the conformational ensembles populated by the disordered polymers in solution. Single-molecule approaches offer information on dynamic and heterogeneous ensembles, capturing distinct and less populated states, overcoming the limitations of average parameter assessment, intrinsic to bulk methods [5–8].

Usually employed in imaging mode [9,10], atomic force microscopy (AFM) can be used in single-molecule force spectroscopy (SMFS) to characterize the statistical distribution of distinct protein conformers in solution. Indeed, protein unfolding under the action of a pulling force has been demonstrated to characterize the molecular structure of tens of distinct proteins and to distinguish among different conformations induced by ligand binding or mutations [11–13]. In the case of the human, amyloidogenic IDP α-synuclein (AS), at least three major conformational states can be recognized [14–16]: random coil (RC), collapsed states stabilized by weak interactions (WI), and compact conformations stabilized by strong interactions (SI). The SMFS technique has been applied to explore the conformational space populated by the different structures of the protein, revealing distinct conformers of the molecular ensemble and structural effects of point mutations linked to familial Parkinson's disease [4,14–17].

Pure AS in vitro, in the absence of interactors, is largely unstructured at neutral pH, with a small fraction of the population in collapsed states of different compactness, as revealed by NMR spectroscopy [18] and small angle X-ray scattering [19]. A particularly compact, globular state is populated in vivo, as indicated by in-cell NMR in neuronal and non-neuronal mammalian cell types [20]. Dopamine (DA) and epigallocatechin-3-gallate (EGCG) are known to bind AS and redirect the aggregation pathway toward soluble oligomers with different structure and toxicity [21,22].

Native mass spectrometry (native MS) has developed into a central tool for structural biology [23–26]. The analysis of charge states populated by globular and disordered proteins by native MS has shown effects of denaturants [27], stabilizers [28], metal binding [29], and protein–protein interactions [30], just to mention some examples. The application of native MS to free AS in solution reveals multimodal charge-state distributions (CSDs), which are suggestive of a conformational ensemble populated by different conformers, in line with the above-mentioned, in vitro and in vivo evidence [29,31–33]. The charge states obtained by proteins in electrospray have long been recognized as affected by protein compactness at the moment of transfer from solution to gas phase [27,34]. This effect can be rationalized by an influence of protein structure on solvent-accessible surface area [35–37] and apparent gas-phase basicity [38].

A large amount of evidence suggests that the ionization patterns of globular and disordered proteins are similarly affected by conformational properties [23,39–41]. Native MS has described conformational responses of AS to alcohols, pH, and copper binding consistent with NMR and other solution methods [29,33]. Native MS has also suggested that binding of DA and EGCG have distinct structural effects on AS soluble monomers [42,43]. While DA preferentially binds and stabilizes an intermediate form, EGCG promotes accumulation of the most compact AS conformer [42]. This different conformational selectivity could help rationalizing the different structure and toxicity of the resulting oligomers, although the two ligands have similar fibrillation-inhibition effects [42]. Nonetheless, the difficulty to capture IDP compact states by small-angle X-ray scattering and ensemble-optimization method has led to the hypothesis that IDP bimodal CSDs are artifacts resulting from a bifurcated ESI mechanism, rather than distinct components reflecting structural heterogeneity of the original protein sample [44]. The aim of this work is to describe AS conformational ensemble and its response to ligands by orthogonal and highly sensitive biophysical techniques, such as SMFS, in order to test the effect of ligand binding in solution and help interpretation of the available native-MS data on AS and IDPs in general. It is found that, while spectroscopic methods sensitive to secondary structure do not capture these conformational transitions, SMFS and native MS reveal rearrangements of the conformational ensembles, consistent with a loss of structural disorder induced by the ligands.

### **2. Results**
