Two-Photon and Time-Resolved Fluorescence Conformational Studies of Aggregation in Amyloid Peptides

The conformational changes associated with the aggregation of proteins are critical to the understanding of fundamental molecular events involved in early processes of neurodegenerative diseases. A detailed investigation of these processes requires the development of new approaches that allow for sensitive measurements of protein interactions. In this paper, we applied two-photon spectroscopy coupled with time-resolved fluorescence measurements to analyze amyloid peptide interactions through aggregation-dependent concentration effects. Labeled amyloid-beta peptide (TAMRA-A beta 1-42) was used in our investigation, and measurements of two-photon-excited fluorescence of the free and covalently conjugated peptide structure were carried out. The peptide secondary structure was correlated with a short fluorescence lifetime component, and this was associated with intramolecular interactions. Comparison of the fractional occupancy of the fluorescence lifetime measured at different excitation modes demonstrates the high sensitivity of the two-photon method in comparison to one-photon excitation (OPE). These results give strong justification for the development of fluorescence-lifetime-based multiphoton imaging and assays.