Saurabh Rai

The study of protein behavior in water-dimethyl sulfoxide (DMSO) solvent mixture is of great interest in biophysical research, as DMSO is a widely used cosolvent in various experimental assays. However, investigating the behavior of intrinsically disordered proteins (IDPs) in a water-DMSO binary solvent mixture is still in its infancy. Herein, we present a comprehensive analysis of the behavior of β-casein (βCN), an IDP, in water-DMSO mixtures using a combination of steady-state fluorescence, time-resolved anisotropy, infrared spectroscopy, and fluorescence correlation spectroscopy (FCS), characterizing the protein stability and conformational dynamics. FCS analysis with Alexa-488-labeled βCN, revealed the variations in hydrodynamic radius with varying content of DMSO, signifying constrained mobility and expanded size of the protein. Further, fluorescence self-quenching of the fluorophore (Alexa-488) was analyzed to probe the conformational dynamics of βCN, with changes in the time constant suggesting protein unfolding followed by an assembly at higher DMSO content. These results were corroborated by infrared spectroscopy, where an increase in the amide-I absorption band indicated the formation of intermolecular antiparallel β-sheet aggregates. At higher DMSO concentrations, additional structural features were observed, which were further examined using optical and field emission scanning electron microscopy (FESEM).

Reported herein are long-lived, red-luminescent silver nanoclusters (AgNCs) protected by the small-molecule ligand thiolactic acid, which exhibit exceptional stability (shelf life exceeding three years, photostability ∼100%), water-solubility, and high biocompatibility, making them suitable for diverse applications such as sensing and live-cell imaging. The AgNCs display extremely sensitive (>2% K^-1) temperature-dependent luminescence, monitored by a dual approach of changes in photoluminescence intensity and excited-state lifetime, enabling precise local thermal environment monitoring with a very high-resolution temperature sensing down to subdegree levels (<0.5 K). MTT assay, confocal fluorescence imaging, and fluorescence lifetime imaging microscopy (FLIM) analysis of mammalian cells suggest that the non-cytotoxic AgNCs specifically stain lysosomes in live cells, functioning as an organelle-specific biomarker and providing critical insights into lysosomal dynamics and intracellular temperature fluctuations. The unique properties of these AgNCs, corroborated by detailed mechanistic studies, open new avenues for studying nanoscale subcellular physiology and developing temperature-sensitive diagnostics and preservation strategies.

Liquid–liquid phase separation (LLPS) exhibited by an ordered protein β-lactoglobulin (β-LG) under crowded environment prompts alterations in conformational distribution leading to emergence of new promiscuous (esterase-like) activity.

This work delineates an integrative approach combining spectroscopic and computational studies to decipher the association-induced fluorescence properties of a fluorescent molecular rotor, viz., auramine O (AuO), after interacting with 20-mer duplex DNA having diverse well-matched base pairs. While exploring the scarcely explored sequence-dependent interaction mechanism of AuO and DNA, we observed that DNA could act as a conducive scaffold to the formation of AuO dimer through noncovalent interactions at lower molecular density. The photophysical properties of AuO depend on the nucleotide compositions as described from sequence-dependent shifting in the emission and absorption maxima. Furthermore, we explored such DNA base pair-dependent fluorescence spectral characteristics of AuO toward discriminating the thermodynamically most stable single nucleotide mismatch in a 20-mer sequence. Our results are interesting and could be useful in developing analogues with further enhanced emission properties toward mismatched DNA sequences.