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.
Highlights
The amyloid fibril formation mechanism is a topic of intensive research given its relationship to a wide variety of human diseases that have high economic and social impacts, some of which are severely impairing, such as Alzheimer’s and Parkinson’s diseases
The incubation in amyloidogenic conditions was performed with an approximate labeled-to-unlabeled protein ratio of 1:700
We kept and analyzed only the soluble fraction that remained in solution. This step allowed us to focus on the small, early amyloidogenic aggregates
Summary
The amyloid fibril formation mechanism is a topic of intensive research given its relationship to a wide variety of human diseases that have high economic and social impacts, some of which are severely impairing, such as Alzheimer’s and Parkinson’s diseases. Additional secondary processes such as fibril fragmentation result in increasingly complex kinetics [4] This mechanism has been observed for all proteins involved in amyloidogenic diseases. Single-molecule multiparameter fluorescence detection [27] can be achieved by sorting the single-molecule events according to their fluorescence intensity, lifetime, and anisotropy This realization requires new technical and methodological advancements that enhance the application of the single-molecule techniques in biophysics. One of these advances is the merger of single-molecule fluorescence spectroscopy with time-correlated single-photon counting techniques using pulsed lasers and appropriate single-photon detectors and electronics. This work demonstrates how these developments in single-molecule techniques can solve relevant biophysical problems
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