Structural and dynamic insights into human disease-related proteins via biophysical approach

Proteins associated with human disease often populate highly dynamic conformational ensembles in which subtle sequence differences and internal motions can strongly influence either normal function or pathological misfolding. This dissertation examines how sequence-encoded dynamics shape molecular behavior in two biologically and clinically important systems: the synuclein family, whose aggregation is linked to Parkinson’s disease, and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates viral entry into host cells. Using an integrated biophysical framework centered on nuclear magnetic resonance (NMR) spectroscopy, this work reveals how conformations of proteins govern their properties and functions.In the first part, the evolutionary origin of aggregation within the synuclein family was investigated by experimentally characterizing reconstructed ancestral proteins. Although modern α-synuclein readily forms amyloid fibrils, its close homologs β- and γ-synuclein show little intrinsic aggregation despite sharing a common domain architecture. Analysis of ancestral synucleins demonstrated that the last common ancestor of the family (ROOT) was intrinsically disordered yet resistant to aggregation, indicating that fibril formation is not an ancestral property. Instead, aggregation competence first emerged at the level of the ancestral αβ-synuclein and was subsequently enhanced along the α-synuclein lineage. Comparisons among ancestral proteins and engineered chimeras established that evolutionary changes within the hydrophobic NAC region were necessary to confer aggregation propensity, while other domains modulate this behavior through transient intramolecular interactions. NMR-based characterization further showed that ROOT adopts a more compact conformational ensemble, in which long-range contacts shield the NAC region, whereas later ancestors and modern α-synuclein populate conformations that more readily expose aggregation-prone segments. These results demonstrate that synuclein aggregation arose through evolutionary reshaping of dynamic intramolecular contacts rather than through the appearance of a single dominant structure.In the second part, the conformational dynamics of the SARS-CoV-2 spike RBD were examined to understand how internal motions contribute to receptor recognition and functional adaptability. Although high-resolution structures have defined the overall fold of the RBD, they provide limited information on the flexibility that enables efficient binding to the host receptor angiotensin-converting enzyme 2 (ACE2) and tolerance to mutation. NMR relaxation analysis revealed that the RBD consists of a relatively rigid β-sheet core accompanied by flexible surface loops and regions undergoing slower conformational exchange, many of which coincide with the receptor binding motif (RBM) and sites frequently mutated in viral variants. These dynamic features indicate that the RBD samples a range of conformations in the solution, providing a structural basis for its ability to accommodate sequence variation while maintaining high-affinity receptor interactions.Together, these studies highlight that protein sequence evolution reshapes conformational dynamics, and these dynamic changes, rather than static structures alone, play a central role in determining biological function and dysfunction. In synucleins, evolutionary modulation of transient intramolecular interactions gave rise to pathogenic aggregation, whereas in the SARS-CoV-2 RBD, localized flexibility and conformational exchange control molecular recognition and viral adaptability. By integrating evolutionary reconstruction with NMR-based analysis of structure and dynamics, this dissertation provides a coherent view of how dynamic landscapes govern both protein misfolding in neurodegenerative disease and receptor engagement in viral infection