Specificity of Interactions Between Intrinsically Disordered Proteins and Small Molecules [electronic resource].

9781392328736

Ann Arbor : ProQuest Dissertations & Theses, 2019.

1 online resource (194 p.)

Access is available to the Yale community.

Intrinsically disordered proteins (IDPs) and regions (IDRs) are central to biology and make up a significant portion of the proteome. Their unique ability to flexibly interconvert between multiple secondary structures, or none at all, make them indispensable components of living systems and participants in numerous key proteinprotein interactions. On the other hand, IDPs are also frequently involved in pathological processes that lead to amyloid formation, a hallmark of multiple degenerative disorders such as Alzheimer's disease, type 2 diabetes, and Parkinson's disease. Given the heterogeneous structural and functional behaviors of IDPs, they can often be difficult to characterize compared to globular proteins and can inhabit broad conformational ensembles that are capable of nucleating dynamic oligomeric assemblies. Nevertheless, efforts to better understand IDP folding and aggregation in physiological and pathological environments are essential for deducing the fundamental interactions that drive disorder and can inform small molecule ligands and compounds that modulate their behaviors.In this dissertation, I focused on two members of the IDP family - islet amyloid polypeptide (IAPP) and a-synuclein (aSyn), which are associated with pathological behavior in the context of type 2 diabetes and Parkinson's disease, respectively. However, the aim was to characterize interactions with small molecules that might be representative for a broader spectrum of !DPs and relevant for both physiological and pathological context.I investigated interactions of IDPs with small molecules of synthetic as well as natural origin, with rigid and flexible structures. Employing several biophysical techniques (isothermal titration calorimetry, crystallography, nuclear magnetic resonance and optical spectroscopies), I studied these interactions in diverse environments including solutions, micelles and giant plasma membrane vesicles (GPMVs) so that a wide range of behaviors could be evaluated.My analysis found that aSyn interacts with complex glycans in solution in a way that affects the majority of the protein sequence, but a specific localized patch, surrounding residue H50, is proposed to play the most significant role. Furthermore, the interaction depends on N-terminal acetylation as well as the presence of physiological membranes in the form of GPMVs.Studies on IAPP and its aggregation inhibitors in the presence of a membrane mimic did not reveal specific hot spots for the interactions but identified two promising compounds that affected the protein in a different way. One of the compounds, an oligoquinoline foldamer (referred to here as ADMI 16), was further investigated in the context of various JAPP models such as a fusion construct with a carrier protein or nonaggregating mutant. Partial assignment of the ADM 116 nuclear magnetic resonance spectrum was performed, which made it possible to validate the folded conformation of the small molecule in an aqueous solution. As a result, more detailed structural studies can now be performed on ADM 116 and its structural analogs to reveal how IAPP and other IDPs are stabilized in their presence. Transient IAPP-ADM 116 interactions observed in solution were strongly perturbed in the presence of dodecylphosphochol ine micelles, further emphasizing the susceptibility of the foldamer to its molecular environment, which is an important aspect of targeting IDPs with this class of small molecules.Results from these combined studies suggest that precisely characterizing aggregation-prone intrinsically disordered proteins requires great attention to the experimental approaches and model systems used to study their behaviors. The propensity of physiological IDPs to form dynamic assemblies at mesoscopic length scales, including liquid-liquid phase transitions, necessitates the use of synergistic tools that capture transient interactions at the molecular, mesa- and macroscopic scales, thereby providing extensive cross-validation with one another. As a whole, this work lays the groundwork for the development of more sophisticated IDP-modulating agents and provides a picture of the behavior of disordered proteins in a wide variety of molecular contexts.

Dissertations & Theses @ Yale University.

Books / Online / Dissertations & Theses

English

January 17, 2020

Thesis (Ph.D.)--Yale University, 2019.

Yale University.