Development and adaptation of new methodologies
A diverse array of existing and emerging spectroscopic and microscopic tools is being developed, adapted, and utilized in our lab to study the behavior of IDPs and the mechanism of phase separation and amyloid formation. The fluorescence depolarization by following the picosecond time-resolved fluorescence anisotropy has been one of the most utilized methodologies in our lab. Using this approach, we have been able to discern various essential modes of chain dynamics (local, segmental/backbone dihedral, long-range, global dynamics) of IDPs and their assemblies on the picosecond-nanosecond timescale. Recently, using anisotropy decay measurements, we have been able to probe energy migration via homo-FRET that illuminates the inner workings of a-synuclein amyloids. These unique and sensitive measurements yielded a 2D-proximity correlation map of a large number of intermolecular distances and detected unique, previously unobserved cross-talks between the hetero-terminal, disordered, fuzzy, inter-protofilament interfaces of the parallel-in-register amyloid spines. A relay of transient interactions via dense, dynamic, fuzzy, and mesh-like network enclosing the ordered amyloid-core can modulate the higher-order packing and nanoscale morphology which governs the amyloid polymorphism responsible for distinct strain-specific disease phenotypes in pathological amyloids. We are also involved in developing and adapting nano-biophysical tools, super-resolution near-field scanning optical microscopy, and ultrasensitive Raman spectroscopic techniques to elucidate the inner workings of amyloids and phase-separated liquid droplets.