ABSTRACT
Oligomeric alpha-synuclein (alphaS) is considered to be the potential toxic species responsible for the onset and progression of Parkinson's disease, possibly through the disruption of lipid membranes. Although there is evidence that oligomers contain considerable amounts of secondary structure, more detailed data on the structural characteristics and how these mediate oligomer-lipid binding are critically lacking. This report is, to our knowledge, the first study that aimed to address the structure of oligomeric alphaS on a more detailed level. We have used tryptophan (Trp) fluorescence spectroscopy to gain insight into the structural features of oligomeric alphaS and the structural basis for oligomer-lipid interactions. Several single Trp mutants of alphaS were used to gain site-specific information about the microenvironments of monomeric alphaS, oligomeric alphaS and lipid-bound oligomeric alphaS. Acrylamide quenching and spectral analyses indicate that the Trp residues are considerably more solvent protected in the oligomeric form compared with the monomeric protein. In the oligomers, the negatively charged C-terminus was the most solvent exposed part of the protein. Upon lipid binding, a blue shift in fluorescence was observed for alphaS mutants where the Trp is located within the N-terminal region. These results suggest that, as in the case of monomeric alphaS, the N-terminus is critical in determining oligomer-lipid binding.
Subject(s)
Fluorescence , Tryptophan/analysis , alpha-Synuclein/chemistry , Amino Acid Substitution/genetics , Lipid Metabolism , Mutagenesis, Site-Directed , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, Tertiary , Spectrum Analysis/methods , Tryptophan/geneticsABSTRACT
We have developed a two-LED wobbler system to generate the spatial displacement of total light intensity on a detector surface, facilitating the acquisition of frequency responses up to 600 kHz with high accuracy. We have used this setup to characterize the low-pass filtering behavior of silicon-based position detectors for wavelengths above 850 nm by acquiring the frequency responses of several quadrant detectors and position-sensitive detectors as functions of wavelength, applied bias voltage, and total light power. We observed an increase in bandwidth for an increase in applied bias voltage and incident-light intensity. The combined effect of these parameters is strongly dependent on the detector used and has significant implications for the use of these detectors in scanning probe and optical tweezers applications.
