Early Sodium Dodecyl Sulfate Induced Collapse of α-Synuclein Correlates with Its Amyloid Formation

Abstract

The aggregation of α-synuclein (A-syn) has been implicated strongly in Parkinson's disease (PD). In vitro studies established A-syn to be a member of the intrinsically disordered protein (IDP) family. This protein undergoes structural interconversion between an extended and a compact state, and this equilibrium influences the mechanism of its aggregation. A combination of fluorescence resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS) has been used to study the membrane induced conformational reorganization and aggregation of A-syn. Different structural and conformational events, including the early collapse, the formation of the secondary structure, and aggregation have been identified and characterized using FCS and other biophysical methods. In addition, the concentrations of glycerol and urea have been varied to study the effect of solution conditions on the above conformational events. Further, we have extended this study on a number of A-syn mutants, namely, A30P, A53T, and E46K. These mutants are chosen because of their known implications in the disease pathology. The variation of solution conditions and mutational analyses suggest a strong correlation between the extent of early collapse and the onset of aggregation in PD.

Small Molecules Attenuate the Interplay between Conformational Fluctuations, Early Oligomerization and Amyloidosis of Alpha Synuclein

“…The expression, purification and fluorophore labeling (where applicable) of WT and mutant proteins have been carried out using published procedure [20]. TC-AS construct was a generous gift from Prof. Donna Arndt-Jovin (Max Planck Institute for Biophysical Chemistry, Laboratory of Cellular Dynamics).…”

“…For the present FCS experiments, we chose a single cysteine mutant of α-syn (G132C), which was labeled using Alexa488Maleimide (Alexa488). G132C α-syn is considered similar to wild type protein for biophysical studies [19,20]. The Alexa488 labeled G132C protein would be referred as Alexa488Syn from now onwards.…”

“…We introduced a cysteine mutant (G132C) at the c-terminal end of the protein. G132C mutant behaves exactly like wild type [20]. This cysteine mutant was labeled with Alexa488Maleimide (Alexa488) using a published procedure [45].…”

Heme minimizes Parkinson’s disease-associated toxicity by inducing a conformational distortion in the oligomers of alpha-Synuclein

“…In the absence of heme, we observed the distinctive sigmoidal aggregation kinetics (23,24) of α-Syn ( Figure 1A). The ThT data was complimented by direct imaging by negative stain TEM and AFM, which clearly showed the formation of a fibrillar network of α-Syn ( Figure 1B).…”

“…Since wild type α-Syn does not have a cysteine residue, a G132C mutation was introduced for the maleimide labelling. This single cysteine mutant G132C is considered similar to the wild type protein for biophysical studies (23,33). While the dissociation constant (Kd) and number of binding sites on the α-Syn G132C monomer for heme was 590 nM and 1 respectively ( Figure 5A), heme binding to α-Syn was not observed when excess (10 mM) imidazole was present ( Figure S4A, SI), suggesting a probable binding of the heme to a histidine residue in the protein.…”