Synthesis and Characterization of N-Doped Carbon Quantum Dots and its Application for Efficient Delivery of Curcumin in Live Cell.

To improve bioavailability, enhance the solubility and stability of the hydrophobic drug curcumin, nanoparticles such as carbon quantum dots (CQDs) are unique choices. In this study, we present a simple, cost-effective, and eco-friendly method for synthesizing nitrogen-doped carbon quantum dots (N-CQDs) and their application in the efficient delivery of hydrophobic drugs curcumin into live cancer cells. The N-CQDs produced in this study exhibit excellent water solubility, remarkable stability, and high biocompatibility.

Enzyme-Regulated Non-Thermal Fluctuations Enhance Ligand Diffusion and Receptor-Mediated Endocytosis.

Active enzymes during catalyzing chemical reactions, have been found to generate significant mechanical fluctuations, which can influence the dynamics of their surroundings. These phenomena open new avenues for controlling mass transport in complex and dynamically inhomogeneous environments through localized chemical reactions. To explore this potential, we studied the uptake of transferrin molecules in retinal pigment epithelium (RPE) cells via clathrin-mediated endocytosis.

Differential Mg deposition on DNA Holliday Junctions dictates the rate and stability of conformational exchange.

DNA Holliday junctions (HJs) are crucial intermediates in genetic recombination and genome repair processes, characterized by a dynamic nature and transitioning among multiple conformations on the timescale ranging from sub-milliseconds to seconds. Although the influence of ions on HJ dynamics has been extensively studied, precise quantification of the thermodynamic feasibility of transitions and detailed kinetic cooperativity remain unexplored. Understanding the heterogeneity of stochastic gene recombination using ensemble-averaged experimental techniques is extremely difficult because of its lack of ability to differentiate dynamics and function in a high spatiotemporal resolution.

Drug Binding to Partially Unfolded Serum Albumin: Insights into Nonsteroidal Anti-Inflammatory Drug Naproxen-BSA Interactions from Spectroscopic and MD Simulation Studies.

Understanding the binding details of a small-molecule drug to a protein in its partially unfolded state is important for drug delivery as it provides insight into the overall drug-binding ability of the protein, even when the majority of binding pockets in its unfolded state are impaired. The interaction of partially unfolded proteins with drugs remains poorly understood due to a lack of structural information on proteins in their partially unfolded states. Here, we studied the interaction between serum albumin (bovine serum albumin (BSA) as a model system), an abundant protein in blood serum that is an effective carrier for numerous known drugs, and a nonsteroidal anti-inflammatory drug (NSAID) naproxen (NPS) using various spectroscopic and computational methods.

Unraveling the Interaction of Diflunisal with Cyclodextrin and Lysozyme by Fluorescence Spectroscopy.

Understanding the interaction between the drug:carrier complex and protein is essential for the development of a new drug-delivery system. However, the majority of reports are based on an understanding of interactions between the drug and protein. Here, we present our findings on the interaction of the anti-inflammatory drug diflunisal with the drug carrier cyclodextrin (CD) and the protein lysozyme, utilizing steady-state and time-resolved fluorescence spectroscopy.

An Innate Host Defense Protein β-Microglobulin Keeps a Check on α-Synuclein amyloid Assembly: Implications in Parkinson's Disease.

Amyloid formation due to protein misfolding has gained significant attention due to its association with neurodegenerative diseases. α-Synuclein (α-syn) is one such protein that undergoes a profound conformational switch to form higher order cross-β-sheet structures, resulting in amyloid formation, which is linked to the pathophysiology of Parkinson's disease (PD). The present status of research on α-syn aggregation and PD reveals that the disease progression may be linked with many other diseases, such as kidney-related disorders.

Interaction of Ibuprofen with Partially Unfolded Bovine Serum Albumin in the Presence of Ionic Micelles and Oligosaccharides at Different λ and pH: A Spectroscopic Analysis.

The interaction between the plasma protein bovine serum albumin (BSA) and the drug ibuprofen (IBU) has been investigated at three different pH values (7.4, 6.5, and 8.

Molecular Mechanism of Interaction of Curcumin with BSA, Surfactants and Live E. Coli Cell Membrane Revealed by Fluorescence Spectroscopy and Confocal Microscopy.

Clinical trials on the therapeutic effect of curcumin have proven to be highly effective against many diseases including cancer and Alzheimer's. However, the molecular mechanism of interaction of curcumin with protein and live cell membrane is poorly understood. Here, we report the mechanism of interaction of curcumin with bovine serum albumin (BSA) and live E.

TCR-pMHC bond conformation controls TCR ligand discrimination.

A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR-pMHC bonds and the conformations of individual TCR-CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR-pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond.

Cathepsin-Mediated Cleavage of Peptides from Peptide Amphiphiles Leads to Enhanced Intracellular Peptide Accumulation.

Peptides synthesized in the likeness of their native interaction domain(s) are natural choices to target protein-protein interactions (PPIs) due to their fidelity of orthostatic contact points between binding partners. Despite therapeutic promise, intracellular delivery of biofunctional peptides at concentrations necessary for efficacy remains a formidable challenge. Peptide amphiphiles (PAs) provide a facile method of intracellular delivery and stabilization of bioactive peptides.

T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition.

Programmed cell death-1 (PD-1) is a coinhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). By titrating PD-1 signaling in a biochemical reconstitution system, we demonstrate that the co-receptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1-recruited Shp2 phosphatase.

Single-molecule fluorescence resonance energy transfer in molecular biology.

Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique for studying the conformation dynamics and interactions of individual biomolecules. In this review, we describe the concept and principle of smFRET, illustrate general instrumentation and microscopy settings for experiments, and discuss the methods and algorithms for data analysis. Subsequently, we review applications of smFRET in protein conformational changes, ion channel open-close properties, receptor-ligand interactions, nucleic acid structure regulation, vesicle fusion, and force induced conformational dynamics.

Single-Molecule Patch-Clamp FRET Anisotropy Microscopy Studies of NMDA Receptor Ion Channel Activation and Deactivation under Agonist Ligand Binding in Living Cells.

N-methyl-d-aspartate (NMDA) receptor ion channel is activated by the binding of two pairs of glycine and glutamate along with the application of action potential. Binding and unbinding of ligands changes its conformation that plays a critical role in the open-close activities of NMDA receptor. Conformation states and their dynamics due to ligand binding are extremely difficult to characterize either by conventional ensemble experiments or single-channel electrophysiology method.

Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state.

Conformational dynamics plays a critical role in the activation, deactivation, and open-close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording.

Solvation dynamics of biological water in a single live cell under a confocal microscope.

Time-resolved confocal microscopy has been applied to study the cytoplasm and nucleus region of a single live Chinese hamster ovary (CHO) cell. To select the cytoplasm and the nucleus region, two different fluorescent probes are used. A hydrophobic fluorescent dye, DCM, localizes preferentially in the cytoplasm region of a CHO cell.

Deoxycholate induced tetramer of αA-crystallin and sites of phosphorylation: fluorescence correlation spectroscopy and femtosecond solvation dynamics.

Structure and dynamics of acrylodan labeled αA-crystallin tetramer formed in the presence of a bile salt (sodium deoxycholate, NaDC) has been studied using fluorescence correlation spectroscopy (FCS) and femtosecond up-conversion techniques. Using FCS it is shown that, the diffusion constant (D(t)) of the αA-crystallin oligomer (mass ~800 kDa) increases from ~35 μm(2) s(-1) to ~68 μm(2) s(-1). This corresponds to a decrease in hydrodynamic radius (r(h)) from ~6.

An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.

The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases.

Femtosecond study of ultrafast fluorescence resonance energy transfer in a catanionic vesicle.

Ultrafast fluorescence resonance energy transfer (FRET) in a catanionic [sodium dodecyl sulfate (SDS)-dodecyltrimethyl ammonium bromide (DTAB)] vesicle is studied by femtosecond up-conversion. The vesicles (diameter ∼400 nm for SDS-rich and ∼250 nm for DTAB-rich vesicles) are much larger than the SDS and DTAB micelles (diameter ∼4 nm). In both micelle and vesicles, FRET occurs in multiple time scales and the time scales of FRET correspond to a donor-acceptor distance varying between 12 and 36 Å.

Diffusion of organic dyes in ionic liquid and giant micron sized ionic liquid mixed micelle: fluorescence correlation spectroscopy.

Diffusion of organic dyes in neat room temperature ionic liquid (RTIL) and RTIL-mixed micelle has been studied by fluorescence correlation spectroscopy (FCS). We have selected two RTILs, 3-pentyl-1-methyl imidazolium bromide ([C5C1Im][Br]) and the corresponding tetra-fluoroborate ([C5C1Im][BF(4)]). Diffusion coefficients (D(t)) of three organic dyes--DCM (neutral), C480 (neutral), and C343 (anionic)--in these RTILs are ∼100 times slower compared to water.

Excited state proton transfer in ionic liquid mixed micelles.

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyranine-1,3,6-trisulfonate, HPTS) in room temperature ionic liquid (RTIL) mixed micelles is studied by femtosecond up-conversion. The mixed micelle consists of a triblock copolymer, (PEO)(20)-(PPO)(70)-(PEO)(20) (Pluronic P123), and one of the two RTILs, 1-pentyl-3-methyl-imidazolium bromide ([pmim][Br]) and 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF(4)]). The size and structure of the mixed micelle vary with the relative amount of the RTIL.

Deuterium isotope effect on femtosecond solvation dynamics in an ionic liquid microemulsion: an excitation wavelength dependence study.

The deuterium isotope effect on the solvation dynamics and the anisotropy decay of coumarin 480 (C480) in a room temperature ionic liquid (RTIL) microemulsion is studied by femtosecond up-conversion. The microemulsion consists of the RTIL 1-pentyl-3-methyl-imidazolium tetra-fluoroborate ([pmim][BF(4)]) in triton X-100 (TX-100)/benzene. Replacement of H(2)O by D(2)O in the microemulsion causes retardation of solvation dynamics.

Deuterium isotope effect on femtosecond solvation dynamics in methyl beta-cyclodextrins.

Deuterium isotope effect on the solvation dynamics and fluorescence anisotropy decay of coumarin 153 (C153) bound to dimethyl beta-cyclodextrin (DMB) and trimethyl beta-cyclodextrin (TMB) is studied using femtosecond upconversion. In D(2)O, there is a marked increase in the steady state emission quantum yield and fluorescence lifetime of C153 bound to DMB and TMB. This suggests strong coupling between C153 and D(2)O inside the cyclodextrin cavity.

Ultrafast FRET in a room temperature ionic liquid microemulsion: a femtosecond excitation wavelength dependence study.

Fluorescence resonance energy transfer (FRET) from coumarin 480 (C480) to rhodamine 6G (R6G) is studied in a room temperature ionic liquid (RTIL) microemulsion by picosecond and femtosecond emission spectroscopy. The microemulsion is comprised of the RTIL 1-pentyl-3-methylimidazolium tetraflouroborate, [pmim][BF4], in TX-100/ benzene. We have studied the microemulsion with and without water.

Femtosecond solvation dynamics in a micron-sized aggregate of an ionic liquid and P123 triblock copolymer.

Dynamic light scattering studies indicate that addition of a room temperature ionic liquid (RTIL, [pmim][Br]), to a triblock copolymer (P123) micelle leads to the formation of giant P123-RTIL clusters of size (diameter) 40 nm in 0.9 M and 3500 nm (3.5 microm) in 3 M RTIL.

A femtosecond study of solvation dynamics and anisotropy decay in a catanionic vesicle: excitation-wavelength dependence.

The structure and dynamics of a catanionic vesicle are studied by means of femtosecond up-conversion and dynamic light scattering (DLS). The catanionic vesicle is composed of dodecyl-trimethyl-ammonium bromide (DTAB) and sodium dodecyl sulphate (SDS). The DLS data suggest that 90 % of the vesicles have a diameter of about 400 nm, whereas the diameter of the other 10 % is about 50 nm.