Fluorescent Probes and Protein Misfolding: Methods and Applications

Abstract: Protein misfolding is a frequently occurring event in living cells and without the quality control, the consequences are disasterous. In this thesis, we studied protein misfolding in vitro by employing a variety of fluorophores, to either detect the highly ordered aggregates of intrinsically disordered proteins/peptides or slight misfolding of a natively folded protein.
Misfolding and concurrent self-assembly of amyloid-beta (Aβ) peptide is believed to be the cause of Alzheimer’s disease. Kinetics of fibril formation correlates with the aggressiveness of a particular variant of Aβ peptide. Hence following the fibril formation in vitro provides a valuable information about the intrinsic and extrinsic factors that play role in the disease development. The most common way of following the amyloid fibril formation in vitro is ThT assay. ThT is a non-covalent fluorescent probe that mainly has affinity for β-sheets. However, unspecific ThT binding to small molecules poses a problem. Here, we developed an alternative assay for ThT, where the mixed aggregates of fluorophore-labeled and unlabeled Aβ are discriminated by their size using a filter-trap and subsequently detected. Signature amyloid fibril formation curves were successfully obtained using filter-trap method, containing information about different stages of fibril formation. We benchmarked the method with two ThT-interfering substances, human serum albumin and negatively charged nanoparticles.
α-synuclein is also an intrinsically disordered protein, playing a central role in Parkinson’s disease pathology. Here, we followed the fibril formation kinetics with ANS, which has affinity to the hydrophobic surfaces on protein, in addition to ThT. ANS reproduces the signature sigmoidal curve, even though it does not have affinity to β-sheets, but hydrophobic patches. This is an implication pointing out that stacking β-sheets and exposure of the hydrophobic patches happen simultaneously. In addition, we investigated the effect of negatively charged vesicles and found that they accelerate the aggregate formation and the surface properties of the aggregates in ratio (lipid/protein) dependent manner. Moreover, lipids were found to be taken up during α-synuclein aggregation up to a certain L/P ca.14.
Valuable information about the hot-spots in the amyloid peptide sequence is obtained via model- and disease related mutations. In second paper, we designed two single mutations for Aβ40 and Aβ42 that contained substitutions from lysine to arginine. We assessed the kinetic effect, microscopic rate constants, structural compatibility by coaggregation and cross-seeding (with wild-type) of these mutated variants as well as the morphology of formed fibrils. We found that Lys-Arg substitution retards the fibril formation in both positions (K16R and K28R). K16R mutation was more effective than K28R, however both mutants shared a common selective hinderance mechanism; a much slower elongation rate. We also found that the wild-type and the mutants are fully compatible with each other in coaggregation and cross-seeding studies and the fibril morphology changes for K28R mutation.
Nanoparticles also induce protein misfolding. In the first study of the second part of the thesis, we developed a method that points the interacting nanoparticle-protein partners. The method employs solvatochromic dyes, as they have affinity to hydrophobic patches, therefore the changes in the intensity with respect to the controls can be correlated with protein misfolding. The method is fully capable of reporting the changes over a time period from miliseconds to days. Moreover, the colloidal stability may also be monitored.
Finally, we invesitaged the protein adsorption to nanoparticles in-depth by partly using the screening method developed. Here, we brought together the most important concepts that play role in protein adsorption (i.e., protein stability, nanoparticle size and surface chemistry) and developed a simple model for the adsorption and misfolding kinetics of proteins on the surface with an emphasis on the dynamics of protein misfolding at different surfaces.