Parkinson's disease in the spotlight : unraveling nanoscale alpha-synuclein oligomers using ultrasensitive single-molecule spectroscopy

Abstract

Niels ZijlstraParkinson's disease in the spotlight Protein folding is an extraordinarily complex process of which the details are still unclear, but is thought to be governed by the energy landscape of the protein and involves a stochastic sampling of many different folds, or conformations, until the protein reaches its thermodynamically most favorable fold [7]. This fold is usually called the native structure of the protein and is mainly determined by the amino acid sequence of the protein [8,9], although the complex environment found in a cell is thought to influence the folding as well [10].The folding of a protein is in some cases co-translational, that is, the folding takes place during synthesis [7,11,12]. In other cases, however, the protein folds in the cytoplasm after a complete synthesis, or is first transported to specific parts of the cell, such as mitochondria or the endoplasmic reticulum, before it folds, see figure 1.1 [7,12].Given the complexity of protein folding and the enormous number of folding events in a cell, it is not surprising that sometimes a protein does not fold correctly or that a protein does not stay folded correctly [1,13]. The protein can get trapped in a local energy minimum resulting in the protein to misfold making the protein toxic [1,14].The more complex the protein folding pathway is, the more likely it is that the folding goes wrong. Normally, a cell can minimize the amount of misfolded proteins by using specialized molecular chaperones or folding catalysts to assist in the folding [4,13]. If the folding still goes wrong, the cell can get rid of the misfolded proteins by degrading them via the ubiquitin-proteasome system, see figure 1.1 [15]. Unfortunately, this defense mechanism does not always work properly. 6 1 1.2. Amyloid diseases and the oligomeric species Amyloid diseases and the oligomeric speciesProtein misfolding and the subsequent aggregation is considered to play an important role in many human neurodegenerative diseases, such as Parkinson's, Alzheimer's, and Huntington's disease, type II diabetes, and in the prion diseases [16]. These diseases are associated with the formation of inter-and intracellular inclusions that mainly contain insoluble amyloid fibrillar aggregates. Amyloid refers to the aggregates having a characteristic cross-β sheet secondary structure. These fibrils are ∼10 nm 7 1 1.2. Amyloid diseases and the oligomeric species in diameter, and can be several microns in length, and thus are composed of many thousands of the constituent monomeric proteins [17]. A commonly used method to detect the formation of amyloid aggregates uses the fluorescent dye Thioflavin T (ThioT). When ThioT binds to β-sheet-rich domains, the dye displays enhanced fluorescence intensity and a characteristic red shift of its emission spectrum. ThioT fluorescence is often used to monitor the kinetics of amyloid formation, which, in general, follows a sigmoidal growth curve that is characterized by an initial lag phase, followed by an exponential growth until it reaches a ...

Fluorescence Methods for Unraveling Oligomeric Amyloid Intermediates

“…For the measurement procedure presented in this chapter, we used a custom-built apparatus (see Fig. 1) (for more details, see [22]). The main components are indicated in the figure, but in general high quality components should be used.…”