Early Amyloidogenic Oligomerization Studied through Fluorescence Lifetime Correlation Spectroscopy

Abstract

Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.

A flash in the pan: Dissecting dynamic amyloid intermediates using fluorescence

“…A variety of modern fluorescence approaches, including fluorescence correlation spectroscopy, fluorescence fluctuation spectroscopy and two-color coincidence detection (TCCD) have been successfully employed to assess the stoichiometry of oligomeric intermediates in amyloidogenic pathways [111][112][113][114][115][116][117][118]. However, obtaining detailed structural insight into these larger species remains challenging.…”

The First Step of Amyloidogenic Aggregation

“…Additional knowledge on the population of these aggregates has been acquired using alternative approaches, which allow for a more complete characterization than classical techniques. For example, dynamic light scattering (DLS) 7 and fluorescence correlation spectroscopy 8 provide insights into the distributions of species based on the diffusional properties. Imaging techniques, such as atomic force microscopy, 9 total internal reflection fluorescence microscopy, 10 and superresolution fluorescence microscopy, 11 have been recently employed to directly probe the shape of aggregates, monitor fibril growth, and directly visualize the localization of aggregates within live cells.…”

“…The addition of the six-residue tag at the C-terminus does not appreciably alter the ability of the construct to form amyloid fibrils ( Figure S1 in Supporting Information). 8 Labeling of the protein at the terminal Cys residue with maleimide-reactive forms of ATTO dyes was performed according to the manufacturer's instructions (see Supporting Information). The N47A-SH3-A488 (labeled with ATTO 488, ATTO-TEC GmbH, Germany) and N47A-SH3-A647N (labeled with ATTO 647N, ATTO-TEC GmbH) samples were purified and lyophilized for storage.…”

Simultaneous measurement of a range of particle sizes during Aβ1–42 fibrillogenesis quantified using fluorescence correlation spectroscopy

“…Despite intense efforts there are currently no validated, reliable and sensitive means to detect Ab oligomers in CSF. The oligomerization and subsequent formation of protein fibrils of Ab is a complex interplay of many peptides and mechanisms and is not yet fully understood [16][17][18][19][20]. However, the majority of studies find that the mechanism for fibrillogenesis is consistent with a nucleation-dependent polymerization model [5,18,21] as illustrated in Fig.…”

“…Several methods have been used to map the progression of peptide aggregation, from monomer to fibril, including, high performance liquid chromatography, gel electrophoresis, atomic force microscopy, transmission electron microscopy (TEM), Thioflavin T assays, dynamic light scattering and others [16]. Monomeric Ab has a hydrodynamic radius (R h ) of 0.9 ± 0.1 nm [23], while oligomeric and fibril forms range in size from several nanometers to several microns [16][17][18][19].…”