Parkinson’s disease (PD) is a currently incurable neurodegenerative disease with motor and non-motor symptoms that impacts the patients’ everyday function and severely affects their quality of life. Current treatments are symptomatic and do not target the cause and molecular mechanism of the disease, which remains elusive. Evidence points towards the aggregation of a small presynaptic protein, alpha-synuclein (aSyn), from its functional disordered form into amyloid fibrils with characteristic β-sheet structure, as being at the centre of PD. However, many unanswered questions remain with regards to the aSyn aggregation mechanism in neurons and, in particular, the earliest steps of this process, when the soluble, functional form of monomeric aSyn misfolds. The motivation of the current thesis is the understanding of the triggers for misfolding, and the structural transitions from the monomer to the fibrillar level in different cellular environmental conditions, to aid the better design of therapeutics in the future. To this aim, different environmental conditions were interrogated with regards to aSyn aggregation propensity, monomer conformation, and amyloid fibril structure. This was approached by studying the aggregation of aSyn in vitro in three levels, with a combination of orthogonal biophysical assays: the bulk aggregation kinetics were monitored by a series of fluorescence-based kinetic assays, the monomeric conformation of aSyn was analysed via Hydrogen-Deuterium Mass Spectrometry (HDX-MS) and the fibril products formed at the end of the kinetic assays were analysed structurally by Atomic Force Microscopy (AFM). The environmental parameters interrogated were high calcium concentrations, (which are particularly relevant in the context of PD pathology), the salts NaCl and KCl (which are the principal ion components of the extracellular and intracellular space, respectively), reduced pH 4 (which roughly corresponds to the lysosomal compartment), building up to a combination of the above to mimic three cellular compartments: extracellular, intracellular, and lysosomal. The study was performed for wild type (WT) aSyn and six familial mutants (FM) (A30P, A53T, E46K, A53E, H50Q, G51D), which are implicated in hereditary PD. In parallel, the effects of aSyn-lipid association on the lipid membranes, and in particular mitochondria were studied to determine whether aSyn brings upon toxicity in additional ways to aggregation, such as mitochondrial dysfunction. This study was performed in vitro with the use of correlative super-resolution optical microscopy (structured illumination microscopy -SIM) and AFM in isolated mitochondria. The first step of this process was the optimisation of the aSyn purification protocol and the production of all the aSyn variants to sufficient amounts and purity. Following that, the combination of cutting edge HDX-MS with established biophysical assays (kinetics, AFM) highlighted a correlation between a shift in the monomeric conformation of aSyn, the protein’s aggregation propensity, and the resulting fibrillar polymorphism. It was established that exposure of the structure to the solvent at the monomer level, particularly at the N-terminus and NAC region, correlates with an increase in aggregation kinetics rates and the formation of more twisted and toxic fibril as the aggregation product. With regards to the environmental conditions, the Ca2+ ion and low pH values were highlighted as an important factor in WT aSyn misfolding, while the monovalent Na+ and K+ ions had a moderate effect on aggregation and the formation of toxic fibril polymorphs. Distinct aggregation kinetics and fibril polymorphs were also observed for the FM of aSyn, pointing towards different monomer conformation distributions and possibly distinct pathways to pathology. With regards to aSyn and lipids, monomeric aSyn in its calcium-bound state was found to reduce the stiffness of mitochondria, possibly further implicating calcium in PD pathology. In this thesis, overall, the identification of aggregation-inducing conditions and the structural characteristics of the monomer and fibrils in said environments is expected to aid in the rational design of therapeutic molecules to prevent aSyn aggregation and PD pathology.