Roles of intermolecular interactions in amyloid fibril formation mechanisms

Amyloid fibrils, a major pathological feature of several neurodegenerative disorders, are highly stable, insoluble aggregates of misfolded proteins. The formation of such aggregates involves a complex equilibrium between protein monomers, different on- and off-pathway transient oligomeric species, and amyloid fibrils. Amyloid fibril formation in vivo may be induced by a myriad of factors, including oxidative stress and alteration of metal ion homeostasis.The work in this thesis involves biophysical studies of the amyloid fibril formation mechanisms of the natively folded Ca2+-binding fish protein, β-parvalbumin (β-PV); and how it is modulated by cell conditions including macromolecular crowding and stabilizing osmolytes, both of which tend to stabilize compact folded protein conformations through an excluded volume- or osmophobic effect, respectively. It was found that when β-PV aggregation is triggered, as occurs upon Ca2+ removal from the protein, spontaneous cystine formation between β-PV monomers initiates the process, whereafter the dimers template monomers into the amyloid conformation, resulting in polymerization. Furthermore, it was discovered that both excluded volume and the osmophobic effect promote the overall aggregation of β-PV, likely by facilitating protein dimerization. Together, these results highlight the potentially detrimental effects of ligand loss and oxidative stress on proteins, whose destabilization might induce amyloid fibril formation that is further exacerbated by otherwise protective in-cell conditions.Amyloid fibril formation by fish β-PV at acidic pH is thought to confer protection against proteolytic degradation in the human gut. In addition, since recent evidence suggests that many incidences of the neurodegenerative disorder Parkinson’s disease (PD) might originate from the gut, a putative interaction between β-PV and α-synuclein (αS), which forms amyloid fibrils in PD, was tested in vitro. Amyloid fibrils of β-PV block αS aggregation, likely by sequestering αS monomers onto the surface, thus potentially implying a protective effect of a diet rich in fish against PD. Lastly, in light of the fact that copper is reduced in affected brain regions of PD patients, as well as the presence of copper binding sites on αS, aggregation of αS in the presence of the endogenous cytoplasmic copper chaperone, Atox1, was studied. It was found that copper-Atox1 interacts with αS and can prevent its aggregation, while apo-Atox1 is ineffective, indicating a copper dependent interaction. The reduced copper levels associated with PD might thus play a role in PD progression by abolishing this protective interaction

Development of bio-mimetic nano-compartments for solar energy capture

A growing range of artificial cell-mimicking compartments(e.g., liposomes) have been demonstrated as technological platforms for applications ranging from model systems in bottom-up cell biology to miniature chemical reactors. Here, I describe work on developing a liposomal compartment for capturing light-energy. The harvesting of light energy starts at a photoactive centre, where light-excited electrons are generated and then transferred to an electron acceptor. The efficiency of this electron transfer is often limited due to charge recombination (i.e., re-assembly of photo-separated electrons and electron holes) within the photoactive chromophore. Inspired by natural photosynthesis, this study envisions a strategy to limit charge recombination by rapid transfer of the lightexcited electrons away from the photoactive molecules (dye-sensitized TiO2 nanoparticles or carbon dots) and across the liposome membrane via conductive transmembrane protein complex MtrCAB from Shewanella oneidensis MR-1. Furthermore, such compartment enables localisation of the oxidation and reduction processes in separate environments. The assembly of the envisioned compartment begins with a study of the molecular interface between TiO2 nanoparticles, a commonly used material for photocatalysis studies, and the MtrC(AB) conduit. This interface is mapped using an approach called protein footprinting, which involves protein labelling and subsequent analysis of the modified peptides by mass spectrometry. Understanding the molecular interactions at this bio-inorganic interface is crucial for engineering electronic communication between these materials. Then, a proof of concept is demonstrated of a half-reaction: light energy capture, charge separation across the membrane and use of the energy to drive a chemical reaction. Transmembrane electron transfer is achieved chemically and photochemically using dye sensitized TiO2 nanoparticles or carbon dots located outside the liposomes. The electron transfer through MtrCAB conduit is confirmed optically by monitoring the destructive reduction of an encapsulated azo-dye Reactive Red 120. Finally, work on encapsulation of fuel evolving catalysts (i.e., hydrogen producing Pt nanoparticles and a hydrogenase HydA1) within the lipid-enclosed compartment (i.e., liposome lumen and porous silica support) is discussed alongside the challenges for combining different materials within ordered structures

PAS Signaling Mechanisms in Aer and Aer2

PAS domains are widespread signal sensors that share a conserved three-dimensional αβ fold that consists of a central β-sheet flanked by several α- helices. The aerotaxis receptor Aer from Escherichia coli and the Aer2 chemoreceptor from Pseudomonas aeruginosa both contain PAS domains. Aer senses oxygen (O2) indirectly via an FAD cofactor bound to its PAS domain, while Aer2 directly binds O2 to its PAS b-type heme cofactor. The Aer and Aer2 PAS domains both interact with a signal transduction domain known as a HAMP domain. The PAS-HAMP arrangement differs between Aer and Aer2, with Aer- PAS residing adjacent to its HAMP domain, and Aer2-PAS being sandwiched linearly between three N-terminal and two C-terminal HAMP domains. The differences between these PAS-HAMP architectures raise the possibility of two different PAS-HAMP signaling mechanisms: a lateral PAS-HAMP signaling mechanism for Aer, and a linear PAS-HAMP signaling mechanism for Aer2. This dissertation focuses on uncovering the PAS-HAMP transduction mechanisms and clarifying the signaling of conserved residues in Aer and Aer2 PAS. In Aer, I determined that a region on the PAS β-scaffold was sequestered by direct interaction with the HAMP domain. These data support a novel lateral PAS-HAMP arrangement that is crucial for Aer signaling. In Aer2, I demonstrated that unique PAS domain residues are involved in heme-binding, oxygen-binding and PAS signal initiation. My data provide the first functional corroboration of the Aer2 PAS signaling mechanism previously proposed from structure. The work presented in this dissertation demonstrates two variations of PAS-HAMP signaling mechanisms, both involving a global conformational change of the PAS domain that is transmitted from the PAS β-scaffold to the HAMP domain. My Aer and Aer2 studies provide the first direct evidence that HAMP domains can be activated by either linear or lateral interaction with a sensor module. Studying PAS-HAMP signaling mechanisms will help in understanding how sensing domains activate chemosensory systems that are involved in the survival of both commensal and pathogenic bacteria

Mass spectrometry analysis of protein/peptide S-palmitoylation

The dynamic S-palmitoylation regulates many intracellular events, including protein trafficking, anchoring, targeting, and protein-protein interactions. Direct detection of S-palmitoylation by conventional liquid chromatography-mass spectrometry (LC-MS) methods is challenging because of the tendency of palmitoyl loss during sample preparation and gas phase fragmentation. Additionally, the high hydrophobicity of the palmitoyl group can prevent proper elution of palmitoyl peptides from the commonly used C18 column. Here, we developed a comprehensive strategy tailored for S-palmitoyl detection using three palmitoyl peptide standards. We found that S-palmitoylation was largely preserved in neutral Tris buffer with tris(2-carboxyethyl)phosphine as the reducing agent and that various fragmentation methods provided complementary information for palmitoyl localization. Moreover, S-palmitoyl peptides were efficiently analyzed using a C4 column and the derivatization of free cysteine with a hydrophobic tag allowed relative quantification of palmitoyl peptides and their unmodified counterparts. We further discovered potential complications to S-palmitoylation analysis caused by the use of ProteaseMAXTM, an MS-compatible detergent. The hydrophobic degradation products of ProteaseMAXTM reacted with the free cysteine thiols, generating artifacts that mimic S-acylation and hydroxyfarnesylation. Another MS-compatible detergent, RapiGestTM, did not produce such artifacts, and showed the ability to stabilize S-palmitoylation by preventing thioester hydrolysis and dithiothreitol-induced thioester cleavage. Moreover, we found that the palmitoyl peptide GCpalmLGNAK could undergo intermolecular palmitoyl migration from the cysteine to the peptide N-terminus or the lysine side chain during sample preparation, and this could lead to false discovery of N-palmitoylation. RapiGestTM inhibited such migration, and is thus recommended for S-palmitoyl sample preparation. We then applied the established method to analyze the regulator of G-protein signaling 4 (RGS4) which had been reported to undergo S-palmitoylation by radioactive labeling. It had also been reported that the S-palmitoylation state of RGS4 affects its GTPase activity. With LC-MS/MS analysis, we found that the addition of palmitate to the cell culture medium in metabolic labeling experiments could boost the level of S-palmitoylation, leading to false discovery of new S-palmitoylation site(s). We also noted discrepancies between the S-palmitoylation sites identified by radioactive labeling and by LC-MS/MS analysis. Further studies are needed to evaluate the reliability of S-palmitoyl detection by these two methods