Investigating the intra-molecular and inter-molecular effects of post-translational modifications on intrinsically disordered protein regions and structured protein regions

Post-translational modifications (PTMs) play a pivotal role in regulating protein structure, interaction, and function. Aberrant PTM patterns are associated with diseases. Moreover, individual PTMs have a complex interaction with each other, known as PTM crosstalk. In the initial two chapters, we combined biophysical experiments and molecular dynamics simulation to study Huntingtin protein (HTT), whose PTMs within the first 17 amino acids (Nt17) influence the conformation, membrane interaction, aggregation and toxicity. We show that oxidation at M8 (oxM8) delays but does not inhibit the aggregation, yet the presence of both oxM8 and acetylation at K6 (AcK6) drastically inhibit mutant HTT exon 1 (mHttex1) fibrillization. PTMs that lower the mHttex1 aggregation rate result in an increased population of a short N-terminal helix in Nt17 or decreased abundance of other helical forms. This underscores the influence of relative abundance of different helical conformation on mHttex1 aggregation, challenging the assumed correlation between overall Nt17 helicity and aggregation. Our results offer new structural perspectives on the differential effects of PTM crosstalks in regulating mHttex1 aggregation.We demonstrate that certain anionic lipids enhance the helical structure of unmodified Nt17. Single acetylation at K6/K9/K15 attenuates such effects, whereas tri-acetylation abolishes Nt17 membrane interaction. Single phosphorylation at S13 and S16 decreases the POPG and PIP2-induced helicity, while dual phosphorylation diminishes Nt17 helicity, regardless of lipid composition. pT3 reduces membrane interactions. oM8 variably affects different membrane-induced helicity in a lipid-dependent way. Our research uncovers the distinct effects of PTM crosstalks on membrane interaction and conformation of Nt17, providing a new understanding of the complex relationship between Nt17 structure, PTMs and membrane binding.Our comprehensive analysis of 1.4 million PTMs with structure and interface prediction tools identified 100k PTMs at various potential intermolecular interfaces and spotlighted PTM 'hotspots' at interfaces as potential regulatory hubs in modulating molecular interactions. Our study enhances the understanding of PTM's regulation of intermolecular interactions, provides a dataset to guide future research, and paves the way for developing computational tools to predict the impact of PTMs on protein interactions.We also explored the limitations and potential of Protein language models (pLMs), which excelled in protein structure prediction. Our research revealed that pLM-based models tend to erroneously predict structures of modified sequences within the context of full-length proteins. We discovered that pLMs make contact predictions based on sequence motifs and their linear distances. Our research advances knowledge of the underlying mechanisms of pLMs, setting the stage for more reliable protein structure predictions and their subsequent use in PTM structural impact prediction. Our integrative analysis sheds light on PTM crosstalk effects on Httex1 conformation, aggregation, and membrane interaction. The work also contributes to a systematic understanding of PTMs at molecular interfaces and evaluates the potential and limitations of current pLMs. This study underscores the intricate mechanisms nature employs to diversify protein function and structure through PTMs, providing insights for future research in this complex field.