Tushar Kanti Mukherjee

Concentration-dependent reversible self-oligomerization of serum albumins through intermolecular β-sheet formation.

Proteins inside a cell remain in highly crowded environments, and this often affects their structure and activity. However, most of the earlier studies involving serum albumins were performed under dilute conditions, which lack biological relevance. The effect of protein-protein interactions on the structure and properties of serum albumins at physiological conditions have not yet been explored. Here, we report for the first time the effect of protein-protein and protein-crowder interactions on the structure and stability of two homologous serum albumins, namely, human serum albumin (HSA) and bovine serum albumin (BSA), at physiological conditions by using spectroscopic techniques and scanning electron microscopy (SEM). Concentration-dependent self-oligomerization and subsequent structural alteration of serum albumins have been explored by means of fluorescence and circular dichroism spectroscopy at pH 7.4. The excitation wavelength (λex) dependence of the intrinsic fluorescence and the corresponding excitation spectra at each emission wavelength indicate the presence of various ground state oligomers of serum albumins in the concentration range 10-150 μM. Circular dichroism and thioflavin T binding assay revealed formation of intermolecular β-sheet rich interfaces at high protein concentration. Excellent correlations have been observed between β-sheet content of both the albumins and fluorescence enhancement of ThT with protein concentrations. SEM images at a concentration of 150 μM revealed large dispersed self-oligomeric states with sizes vary from 330 to 924 nm and 260 to 520 nm for BSA and HSA, respectively. The self-oligomerization of serum albumins is found to be a reversible process; upon dilution, these oligomers dissociate into a native monomeric state. It has also been observed that synthetic macromolecular crowder polyethylene glycol (PEG 200) stabilizes the self-associated state of both the albumins which is contrary to expectations that the macromolecular crowding favors compact native state of proteins.

Photophysical study of a π-stacked β-sheet nanofibril forming peptide bolaamphiphile hydrogel

We describe the state of molecular self-assembly of a peptide based bolaamphiphile molecule using spectroscopic and microscopic techniques. The tryptophan and phenylalanine containing peptide bolaamphiphile forms a hydrogel upon sonication under physiological conditions. Sonication helps to reorient the peptide molecules by providing the required energy for the self-assembly process. The disassembly and self-assembly processes are influenced by various stimuli, including heating–cooling and shaking–rest methods. The extensive hydrogen bonding and π–π stacking interactions are responsible for the self-assembly process, which is confirmed by FT-IR, temperature dependent NMR and fluorescence spectroscopy studies. FT-IR and powder X-ray diffraction studies reveal that the gelator molecules self-assemble into an antiparallel β-sheet type structure. The TEM image of the hydrogel shows a well-defined amyloid-like nanofibrillar structure. The amyloid-like behaviour of the fibril forming peptide bolaamphiphile hydrogel is confirmed by ThT and Congo red binding studies. The effect of concentration, time and temperature on the self-assembly mechanism of the peptide bolaamphiphile hydrogel is investigated by time resolved fluorescence spectroscopy.

Direct Evidence of Intrinsic Blue Fluorescence from Oligomeric Interfaces of Human Serum Albumin

The molecular origin behind the concentration-dependent intrinsic blue fluorescence of human serum albumin (HSA) is not known yet. This unusual blue fluorescence is believed to be a characteristic feature of amyloid-like fibrils of protein/peptide and originates due to the delocalization of peptide bond electrons through the extended hydrogen bond networks of cross-β-sheet structure. Herein, by combining the results of spectroscopy, size exclusion chromatography, native gel electrophoresis, and confocal microscopy, we have shown that the intrinsic blue fluorescence of HSA exclusively originates from oligomeric interfaces devoid of any amyloid-like fibrillar structure. Our study suggests that this low energy fluorescence band is not due to any particular residue/sequence, but rather it is a common feature of self-assembled peptide bonds. The present findings of intrinsic blue fluorescence from oligomeric interfaces pave the way for future applications of this unique visual phenomenon for early stage detection of various protein aggregation related human diseases.

Resonant excitation energy transfer from carbon dots to different sized silver nanoparticles

The influence of size on the efficiency of the nanometal surface energy transfer (NSET) process between excited donors and different sized metal nanoparticles (NPs) is poorly explored in the literature. Here we present a systematic study by correlating the size of silver nanoparticles (Ag NPs) with the efficiency of excitation energy transfer (EET) from carbon dots (CDs) to Ag NPs. Three different sized citrate-capped Ag NPs with a mean hydrodynamic diameter of 39.91 ± 1.03, 53.12 ± 0.31 and 61.84 ± 0.77 nm have been synthesized for the present study. The estimated zeta potential of the synthesized CD is -25.45 ± 1.23 mV while that for the smallest, medium and largest sized Ag NPs are -76.24 ± 3.92, -67.60 ± 4.40, and -58.01 ± 3.10 mV, respectively. The steady-state and time-resolved PL measurements reveal a significant PL quenching of CDs as a function of Ag NP size. A control experiment with Ag NPs having a LSPR at 398 nm shows a negligible amount of PL quenching of CDs as a consequence of inadequate spectral overlap. The origin behind this PL quenching of CDs has been rationalized on the basis of the increased nonradiative decay rate due to NSET from the CDs to the Ag NP surface. Various energy transfer related parameters have been estimated from the NSET theory and it has been observed that the NSET efficiency increases with the increase in the size of Ag NPs. This phenomenon has been explained by considering a larger spectral overlap and a shorter separation distance between the CDs and larger sized Ag NPs due to reduced electrostatic repulsion. Our present results reveal that the size of NPs plays an important role in the NSET process and this phenomenon can be easily utilized to tune the efficiency of energy transfer for various applications.

Synergistic Enhancement of Electron-Accepting and -Donating Ability of Nonconjugated Polymer Nanodot in Micellar Environment

Understanding the fundamental electron-transfer dynamics in photoactive carbon nanoparticles (CNPs) is vitally important for their fruitful application in photovoltaics and photocatalysis. Herein, photoinduced electron transfer (PET) to and from the nonconjugated polymer nanodot (PND), a new class of luminescent CNP, has been investigated in the presence of N,N-dimethylaniline (DMA) and methyl viologen (MV2+) in homogeneous methanol and sodium dodecyl sulfate (SDS) micelles. It has been observed that both DMA and MV2+ interact with the photoexcited PND and quench the PL intensity as well as excited-state lifetime in bulk methanol. While in bulk methanol, purely diffusion-controlled PET from DMA to MV2+ via PND has been observed, the mechanism and dynamics differ significantly in SDS micelles. In contrast to homogeneous methanol medium, a distinct synergic effect has been observed in SDS micelles. The presence of both DMA and MV2+ enhances the electron-accepting and -donating abilities of PND in SDS micelles. Time-resolved photoluminescence (PL) measurements reveal that the PET process in SDS micelles is nondiffusive in nature mainly due to instantaneous electron transfer at the confined micellar surface. These results have been explained on the basis of heterogeneous microenvironments of SDS micelles which compartmentalize the donor and acceptor inside its micellar pseudo phase. The present findings provide valuable insights into the intrinsic relation between redox and PL properties of nonconjugated PND.