Research Overview

Transport of single-molecule (SM) fluorescent tracers provides a wealth of information regarding the local environment of heterogeneous media (doi.org/10.1063/1.5128743). However, the localization error (LE) in SM tracking (SMT) is considerably larger (~30-50 nm) than size of molecular tracers (~1-3 nm), and hence instances of genuine transient stop remain unreliable at molecular length scales in SM trajectories. We propose that authentic pauses within LE can be revealed upon probing SM reorientational dynamics (doi.org/10.1021/acs.jpcb.6b08584) based on the premise that passive tracers' translation is associated with fast dipolar rotation. We demonstrate how polarization-resolved SMT (PR-SMT) can provide emission-anisotropy at each super-localized position, thereby revealing tumbling propensity of SMs during random walks. Our PR-SMT results on rhodamine 6G tracers inside poly(vinylpyrrolidone) thin-film indicate the existence of nanoscale glassy domains in a pool of rubbery polymer networks far above the glass transition (doi.org/10.1039/D0CP05864E). 

Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative including Alzheimer’s, Creutzfeldt–Jakob, Huntington’s, and Parkinson’s diseases. The secondary structure of amyloid fibrils is remarkably uniform composed of characteristic “cross-β” sheets. Polarization-dependent fluorescence response of dyes bound to ordered structures is a powerful and well-established technique to extract structural information for a wide range of systems. Here, the bound Thioflavi T in the amyloid fibril is modeled with conical distribution of dyes enabling us to estimate the fluorescence intensity in the two orthogonal polarized channels (s, p) at arbitrary fibril orientation. The model suggests that the parallel orientation of the cone axis with the s channel yields the maximum anisotropy that solely depends on the cone aperture. As a proof of principle, amyloid fibrils inspired from Aβ(1–42) have been investigated with incremental analyzer rotation to reveal orientation and angular distribution of dye. The variation of cone axis and the angular distribution is linked with the polymorphism of amyloid fibril (doi.org/10.1021/acs.jpcb.1c08604).

A plethora of biological processes hinge on the intricate dance of biomolecular interactions. Typically, scientists use the traditional fluorescence co-localization method to investigate how biomolecules interact in close proximity. However, there's a catch – this method has a resolution limit of around 200nm. In this tiny space, millions of molecules can be crammed together, making it tricky to confirm if the observed signals truly indicate interactions between biomolecules. To address this challenge, we introduce a novel spectrally-resolved fluorescence microscopy technique centered on energy transfer (ET) to validate these biomolecular interactions. The beauty of ET lies in its sensitivity to distance – it significantly changes and becomes negligible after a few molecular dimensions. This allows us to measure sensitized emission, proportionate to the extent of ET, providing a reliable method to explore local interactions among biomolecules labeled with donors and acceptors. Our technique has already shown promising results in practical applications. We successfully demonstrated its effectiveness in studying phase-separated α-synuclein droplets, the local surface polarity of α-Syn fibril and investigating supramolecular block copolymers featuring alternate donor- and acceptor-labeled segments.

The aggregation of synthetic peptides derived from the 21st amino acid, i.e., selenocysteine, has been studied in collaboration with Prof. H.B. Singh. To our surprise, we observed that seleno-peptides forms supramolecular architecture similar to the proteinous aggregates. We demonstrate the first-ever reported self-assembled structures of any selenopeptides. The nanoscale aggregates were found to be amyloid fibrils (doi.org/10.1039/C8CC06528D). In another study, we reported an entirely different class of peptides that self-assembled into a well-ordered mesoscale tubular aggregate. Despite the broad interest of peptide nanotubes, a limited number of peptides have been reported till now. Based on the selenopeptide chemistry, we have demonstrated a new peptide-conjugates for such tubular morphology and deciphered the impact of Se and the heteroatoms (doi.org/10.1021/acsabm.0c01551). Various physicochemical aspects are yet to be deciphered about these new selenopeptides forming different assembled structures. I feel happy to be a part at the beginning. We have explored different techniques and designed experiments beyond our expertise to understand the system.