Conformational Switch Driven Membrane Pore Formation by Mycobacterium Secretory Protein MPT63 Induces Macrophage Cell Death.

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

Development of a near infrared Au-Ag bimetallic nanocluster for ultrasensitive detection of toxic Pb2+ ions in vitro and inside cells.

Although the research activities pertaining to the synthesis of fluorescent noble metal nanoclusters (NCs) and their applications in biological optics have been growing, only limited information is available in the near IR (NIR) region. However, fluorescence spectroscopy and microscopy in the NIR region offer significant advantages over UV and visible wavelengths. In this manuscript, we demonstrate bio-mineralized synthesis of stable Au-Ag bimetallic NCs with tunable NIR fluorescence using bovine serum albumin (BSA) as a protein template. We also demonstrate its application in the detection of toxic heavy metal ions Pb2+ in vitro and inside cells. The tunability of the fluorescence emission between 680 nm and 815 nm is achieved by systematically varying the ratio of Au and Ag in the composite NCs. The bimetallic NCs when interacting with Pb2+ offered a large increase in fluorescence intensity, which enabled sensitive detection of Pb2+. We determined a limit of detection (LOD) of 96 nM for the detection of Pb2+ under in vitro conditions, which is significantly less than the safe level in drinking water. Its applicability has also been demonstrated successfully in real water samples collected from local water bodies.

Protein Fibril-Templated Biomimetic Synthesis of Highly Fluorescent Gold Nanoclusters and Their Applications in Cysteine Sensing.

Biomimetic synthesis of multifunctional fluorescent gold nanoclusters (Au NCs) is of great demand because of their ever-increasing applications. In this study, we have used self-assembled bovine serum albumin (BSA) amyloid-like nanofibers as the bioinspired scaffold for the synthesis of Au NCs. The amyloid fibril stabilized gold nanocluster (Fib-Au NC) has been found to have appreciable enhancement of fluorescence emission and a large 25 nm red shift in its emission maxima when compared to its monomeric protein counterpart (BSA-Au NC). The underlying mechanism accountable for the fluorescence behavior and its spectral shift has been thoroughly investigated by a combined use of spectroscopic and microscopic techniques. We have subsequently demonstrated the use of Fib-Au NCs for cysteine (Cys) sensing both in vitro and inside live cells. Additionally, cellular uptake and postpermeation effect of Fib-Au NCs have also been ascertained by detailed flow cytometry analysis, viability assay, and real-time apoptotic gene expression profiling.

Nanoparticle Induced Conformational Switch Between α-Helix and ß-Sheet Attenuates Immunogenic Response of MPT63.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from ß-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a ß-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

Understanding the Effect of Single Cysteine Mutations on Gold Nanoclusters as Studied by Spectroscopy and Density Functional Theory Modeling.

Fluorescent metal nanoclusters have generated considerable excitement in nanobiotechnology, particularly in the applications of biolabeling, targeted delivery, and biological sensing. The present work is an experimental and computational study that aims to understand the effects of protein environment on the synthesis and electronic properties of gold nanoclusters. MPT63, a drug target of Mycobacterium tuberculosis, was used as the template protein to synthesize, for the first time, gold nanoclusters at a low micromolar concentration of the protein. Two single cysteine mutants of MPT63, namely, MPT63Gly20Cys (mutant I) and MPT63Gly40Cys (mutant II) were employed for this study. The experimental results show that cysteine residues positioned in two different regions of the protein induce varying electronic states of the nanoclusters depending on the surrounding amino acids. A mixture of five-atom and eight-atom clusters was generated for each mutant, and the former was found to be predominant in both cases. Computational studies, including density functional theory (DFT), frontier molecular orbital (FMO), and natural bond orbital (NBO) calculations, validated the experimental observations. The as-prepared protein-stabilized nanoclusters were found to have applications in the imaging of live cells.

Aggregation-induced fabrication of fluorescent organic nanorings: selective biosensing of cysteine and application to molecular logic gate.

Self-aggregation behavior in aqueous medium of four naphthalimide derivatives has exhibited substitution-dependent, unusual, aggregation induced emission enhancement (AIEE) phenomena. Absorption, emission, and time-resolved study initially indicated the formation of J-type fluorescent organic nanoaggregates (FONs). Simultaneous applications of infrared spectroscopy, theoretical studies, and dynamic light scattering (DLS) measurements explored the underlying mechanism of such substitution-selective aggregation of a chloro-naphthalimide organic molecule. Furthermore, transmission electron microscopy (TEM) visually confirmed the formation of ring like FONs with average size of 7.5-9.5 nm. Additionally, naphthalimide FONs also exhibited selective and specific cysteine amino acid sensing property. The specific behavior of NPCl aggregation toward amino acids was also employed as a molecular logic gate in information technology (IT).

Spectroscopic and quantum mechanical approach of solvatochromic immobilization: modulation of electronic structure and excited-state properties of 1,8-naphthalimide derivative.

Photophysical and spectroscopic properties of a fluorescent analogue, 2-(5-selenocyanato-pentyl)-6-chlorobenzo- [de]isoquinoline-1,3-dione (NP) in different solvents has been described in this paper using steady-state, time resolved spectroscopy and density functional theory (DFT) calculation. Stoke's shifted emission band in different solvents clearly demonstrate the highly polar character of the excited state, which is also supported by the enhancement of dipole moment of the molecule upon photoexcitation. Spectroscopic studies and multiple linear regression analysis method reveal that the solvatochromic behavior of the probe depends not only on the polarity of the medium but also on the hydrogen bonding interaction with the solvents. When the solvent effect was taken into account, the computed results show encouraging agreement with known experimental data. This article reveals the excellent correlation between the predicted and experimental spectral data of 1,8-naphthalimide derivative, providing a useful tool in the design of new fluorogenic probes having potential therapeutic activity.

An efficient, Schiff-base derivative for selective fluorescence sensing of Zn²âº ions: quantum chemical calculation appended by real sample application and cell imaging study.

Since zinc ions (Zn(2+)) are involved in numerous biological phenomena and go through subsequent interactions with zinc-binding proteins, we have attempted a sensitive fluorescence based detection of this second most abundant metal ion using an engineered and synthesized Schiff-base ligand, namely 2,4-bis((Z)-2-(1-(pyridin-2-yl)ethylidene)hydrazinyl)pyrimidine (PyHP). The ligand exhibits a zinc-induced fluorescence response when investigated in a MeOH-buffer (10 mM HEPES, pH = 7) (4 : 1) solvent mixture. The presence of zinc ions (λ(ex) = 410 nm, quantum yield, ϕ = 0.20) causes approximately 45 fold fluorescence enhancement at 489 nm. Formation of the metal-ligand complex was ascertained by (1)H NMR and mass spectra analysis. 1 : 1 binding affinity was ascertained according to Job's plot. Apart from this, theoretical interpretation of the experimental outcome was also obtained by applying density functional theory (DFT) to the PyHP-Zn(2+) complex formation. The practical applicability of the ligand has been tested in bacterial cells as well as in mammalian cell imaging and also by measuring and comparing the amount of Zn(2+) in some real samples such as liquid milk, tomato juice, banana stem juice and commercial fruit juice.

Unveiling the groove binding mechanism of a biocompatible naphthalimide-based organoselenocyanate with calf thymus DNA: an "ex vivo" fluorescence imaging application appended by biophysical experiments and molecular docking simulations.

The present study embodies a detailed investigation of the binding modes of a potential anticancer and neuroprotective fluorescent drug, 2-(5-selenocyanato-pentyl)-6-chloro benzo[de]isoquinoline-1,3-dione (NPOS) with calf thymus DNA (ctDNA). Experimental results based on spectroscopy, isothermal calorimetry, electrochemistry aided with DNA-melting, and circular dichroism studies unambiguously established the formation of a groove binding network between the NPOS and ctDNA. Molecular docking analysis ascertained a hydrogen bonding mediated 'A-T rich region of B-DNA' as the preferential docking site for NPOS. The cellular uptake and binding of NPOS with DNA from "Ehrlich Ascites Carcinoma" cells confirmed its biocompatibility within tumor cells. Experimental and ex vivo cell imaging studies vividly signify the importance of NPOS as a potential prerequisite for its use in therapeutic purposes.

Facile room temperature synthesis of lanthanum oxalate nanorods and their interaction with antioxidative naphthalimide derivative.

Polycrystalline lanthanum oxalate (LaOX) nanorods (NRs) were succesfully synthesized using reverse micellar (RM) method. The study espouses the versatility of the reverse micellar method forming monodisperse, stable nanorods at room temperature. The as-synthesized LaOX nanorods were characterised by different techniques. Result shows that the nanorods of LaOX with aspect ratio 10.2:1 have preferred growth direction of (020). The pure phase synthesis of the nanorods was confirmed from Fourier transformed infrared (FTIR) and X-ray diffraction (XRD) analysis. The thermo gravimetric analysis (TGA) and differential thermal analysis (DTA) of the as-synthesized nanorods were perfomed to observe their thermal degradation. Synthesized nanorods are fluorescent having emission maximum at 329 nm. Fluorescence resonance energy transfer (FRET) phenomenon has been observed between LaOX nanorods (energy donor) and naphthalimide (NAP) derivative (energy acceptor) and this mechanism can be helpful for sensing of NAP or NAP like molecules used as antioxidative in living systems.

Differential contribution of Igepal and CnTAB micelles on the photophysics of nonsteroidal drug Naproxen.

Spectroscopic studies of Naproxen (NP), a nonsteroidal drug have been carried out in well characterized, micellar media of cationic surfactants of a homologous series having general formula C(n)TAB (alkyl trimethyl ammonium bromide) and of nonionic surfactants of Igepal (Ig) series (poly(oxyethylene) nonyl phenol). The fluorescence behavior of the drug molecule in C(n)TAB micelles has been found to be opposite to that in Igepal micelles. The binding constants during probe micelle binding have been evaluated from relevant fluorescence data. Location and nature of the surrounding medium of the probe in micellar media have been ascertained from fluorescence quenching study. Fluorescence anisotropy parameter has been monitored for exploring the imposed motional restriction of the microenvironment around the probe. Contrasting behavior of the drug molecule has been observed in two different types of micelles. Based on the experimental and theoretical studies, an attempt has been made to explain the different behavior of the probe in different media.