Partha Pratim Parui

Formation of oligomeric cytochrome c during folding by intermolecular hydrophobic interaction between N- and C-terminal α-helices.

We have previously shown that horse cytochrome c (cyt c) forms oligomers by domain swapping its C-terminal α-helix when interacting with ethanol. Although folding of cyt c has been studied extensively, formation of domain-swapped oligomers of cyt c during folding has never been reported. We found that domain-swapped oligomeric cyt c is produced during refolding from its guanidinium ion-induced unfolded state at high protein concentrations and low temperatures. The obtained dimer exhibited a domain-swapped structure exchanging the C-terminal α-helical region between molecules. The extent of dimer formation decreased significantly for the folding of C-terminal cyt c mutants with reduced hydrophobicity achieved by replacement of hydrophobic residues with Gly in the C-terminal region, whereas a large amount of heterodimers was generated for the folding of a mixture of N- and C-terminal mutants. These results show that cyt c oligomers are formed through intermolecular hydrophobic interaction between the N- and C-terminal α-helices during folding. A slow phase (4-5 s) was observed in addition to a 400-500 ms phase during folding of a high concentration of cyt c in the presence of 1.17 M guanidine hydrochloride. The fast phase is attributed to the intramolecular ligand exchange process, and we attribute the slow phase to the ligand exchange process in oligomers. These results show that it is important to consider formation of domain-swapped oligomeric proteins when folding at high protein concentrations.

A cyanide selective off–on fluorescent chemosensor with in vivo imaging in 100% water: solid probe preferred over in situ generation

A nontoxic fluorescent chemosensor [Cu(BP)HMB]2(ClO4)2 (1) synthesized in solid phase, exhibits unprecedented selectivity and sensitivity over the allied in situ complexes to perform fluorescence in “turn-off–on” mode for sensing cyanide in 100% aqueous medium under physiological conditions and for in vivo imaging using the nematode C. elegans. Below μM detection limit, instantaneous and excellent ratiometric responses are also beneficial to detect trace amounts of anthropogenic or biogenic cyanide.

Formation of Domain-Swapped Oligomer of Cytochrome c from Its Molten Globule State Oligomer

Many proteins, including cytochrome c (cyt c), have been shown to form domain-swapped oligomers, but the factors governing the oligomerization process remain unrevealed. We obtained oligomers of cyt c by refolding cyt c from its acid molten globule state to neutral pH state under high protein and ion concentrations. The amount of oligomeric cyt c obtained depended on the nature of the anion (chaotropic or kosmotropic) in the solution: ClO4(-) (oligomers, 11% ± 2% (heme unit)), SCN(-) (10% ± 2%), I(-) (6% ± 2%), NO3(-) (3% ± 1%), Br(-) (2% ± 1%), Cl(-) (2% ± 1%), and SO4(2-) (3% ± 1%) for refolding of 2 mM cyt c (anion concentration 125 mM). Dimeric cyt c obtained by refolding from the molten globule state exhibited a domain-swapped structure, in which the C-terminal α-helices were exchanged between protomers. According to small-angle X-ray scattering measurements, approximately 25% of the cyt c molecules were dimerized in the molten globule state containing 125 mM ClO4(-). These results indicate that a certain amount of molten globule state oligomers of cyt c convert to domain-swapped oligomers during refolding and that the intermolecular interactions necessary for domain swapping are present in the molten globule state.

A unique cysteine selective water soluble fluorescent probe operable in multiple sensing cycles for the detection of biogenic cysteine in multicellular living species

The Schiff base ligand (L1) showing large fluorescence intensity in an aqueous medium owing to a ground state intramolecular proton transfer (GSIPT) reaction was utilized in a complexation process to generate a highly stable non-fluorescent Cu(II)/L1 (1) in its “turn-off” mode. The 3:2 Cu(II)/L1 stoichiometric complex (1), isolated in the solid form, exhibited a cysteine (Cys)-induced “turn-on” fluorescence response with a unique time-dependent intensity variation. Unlike other biothiols, the partially deprotonated –SH moiety in Cys is highly susceptible for participating in an ionic interaction with Cu(II)-centres in 1 at a physiological pH of ∼7.4, followed by reduction to Cu(I), and then a subsequent demetallation, resulting in a large increment of the fluorescence intensity for free fluorophoric L1, which persists for about 4 –13 min owing to an unexpected stability of the Cu(I) ions. After Cys consumption, Cu(I) ions in aqueous solution rapidly oxidize to participate further in complexation with the existing free L1. The resulting complex may again be susceptible to Cys-induced abovementioned decomplexation followed by complexation for at least four different external Cys additions in four successive cyclic pathways. This multiple Cys sensing behaviour has been exploited to recognize the existence of Cys in multicellular Caenorhabditis elegans (C. elegans).

Planar–rotor architecture based pyrene–vinyl–tetraphenylethylene conjugated systems: photophysical properties and aggregation behavior

Four pyrene-vinyl-tetraphenylethylene based conjugated materials were synthesized and characterized by FT-IR, NMR, and mass spectroscopy. The photophysical (including absorption, fluorescence, and fluorescence lifetime) and aggregation properties in tetrahydrofuran were investigated. The photophysical and aggregation behavior depends on the spacer, substituent, and also the architecture (mono or tetra-branched) of the molecule. The vinyl spacer mono-branched compound is aggregation induced emission (AIE) active (αAIE = ∼6). Vinyl spacer tetra-branched compounds are AIE inactive, but their emitting efficiency is good in both solution (Φfl = 63%) phase and in the aggregated state (Φfl = 43%). Phenylvinyl spacer tetra-branched compounds emit light strongly in solution (Φfl = 92%), but not in the aggregated state (Φfl = 8%). They are shown to be thermally stable and emit light in the green region (500-550 nm). The results of cyclic voltammetry measurements of these materials showed irreversible oxidation waves, and have high HOMO energy levels (-5.66 to -5.53 eV).

Large magnetic field effect on back electron transfer from uncharged radical to its cationic partner in anionic micelle

The magnetic field effect (MFE) on the radical pair (RP) generated by photoexcitation of the acetyl derivative of phenyl pyrylium ion (APP+) in the presence of biphenyl, an electron donor, has been investigated. The escape yield at 3.5 T is more than ten times the zero-field value. The MFE reaches near-saturation twice, once at fields of the order of 10mT and again at about 3.5 T. The low-field variation of the MFE conforms to the pattern expected for the isotropic HFC mechanism, and the high-field variation to that expected for the relaxation mechanism. In this particular system two types of radical pair are generated, one by electron transfer from the donor to the acceptor and another by H-abstraction from the micelle. The MFEs on the two types of 3RP have been compared.

Interfacial pH and polarity detection of amphiphilic self-assemblies using a single Schiff-base molecule

The interfacial pH and polarity are two highly interrelated parameters in amphiphilic self-assembled systems. The hydronium ion (H3O+) concentration and/or the pH value near the water/oil separating interface may change significantly due to large polarity gradients between water- and oil-exposed surfaces within the interface. Therefore, for precise detection of these two properties (pH and polarity) at a specific interfacial depth, a similar interfacial location of the same probe is a prerequisite. In this regard, we have synthesized a new interface-interacting Schiff-base (SBOH-Z-SBOH) molecule to detect both the interfacial pH and polarity of various amphiphilic self-assembled micelles and vesicles at a similar interfacial location. SBOH-Z-SBOH, existing mostly as a non-ionic species (SBOH0-Z-SBOH0) in nonpolar solvents, exhibits an exclusive solvent polarity-dependent linear interconversion equilibrium with its partially charge separated zwitterionic form (SBOH0-Z-SBOH±) as the polarity of the medium increases, which makes it useful to detect the polarity. Additionally, the solvent pH-dependent conversion of both SBOH0-Z-SBOH0 and SBOH0-Z-SBOH± into the deprotonated di-anionic species (SBO−-Z-SBO−) allows it to monitor the pH. We found that the interfacial dielectric constant (∼44.0–54.0) differs substantially from that of the bulk aqueous medium depending on the amphiphilic system. On the other hand, unlike the neutral interface of titron X-100 (TX-100) micelles or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) unilamellar vesicles (ULVs), a large positive pH-deviation of ∼1.8 and 2.2 units from the bulk to the interface was identified for cationic cetrimonium bromide (CTAB) micelles and dimethyldioctadecylammonium bromide (DDAB) ULVs, respectively. The present study provides a unique and simple Schiff-base molecule to monitor the pH and polarity at similar interfacial depths for amphiphilic self-assembled systems.

Exploitation of a new Schiff-base ligand for boric acid fluorescent sensing in aqueous medium with bio-imaging studies in a living plant system

A hydrolysis resistant Schiff-base ligand (L1) consisting of an aromatic aldimine moiety appended to a triazole precursor along with phenolic–OH has been synthesized which acts as a fluorescence probe and exhibits rare selective sensing ability for boric acid (BA) in aqueous buffer medium at pH 7.3 by forming a 1 : 1 chelate complex. Reasons behind the complexation and subsequent fluorescence enhancement are well established using experimental as well as theoretical evidence. Bio-imaging studies are done using Arabidopsis Thaliana as the model plant system.

Detection of Curvature-Radius-Dependent Interfacial pH/Polarity for Amphiphilic Self-Assemblies: Positive versus Negative Curvature

It is possible that a defined curvature at the membrane interface controls its pH/polarity to exhibit specific bioactivity. By utilizing an interface-interacting spiro-rhodamine pH probe and the Schiff base polarity probe, we have shown that the pH deviation from the bulk phase to the interface (ΔpH)/interfacial dielectric constant (κ(i)) for amphiphilic self-assemblies can be regulated by the curvature geometry (positive/negative) and its radius. According to 1H NMR and fluorescence anisotropy investigations, the probes selectively interact with an anionic interfacial Stern layer. The ΔpH/κ(i) values for the Stern layer are estimated by UV-vis absorption and fluorescence studies. For the anionic sodium bis-2-ethylhexyl-sulfosuccinate (AOT) inverted micellar (IM) negative interface, the highly restricted water and proton penetration into the Stern layer owing to tight surfactant packing or a reduced water-exposed headgroup area may be responsible for the much lower ΔpH ≈ -0.45 and κ(i) ≈ 28 in comparison to ∼-2.35 and ∼44, respectively, for the anionic sodium dodecyl sulfate (SDS) micellar positive interface with a close similar Stern layer. With increasing AOT IM water-pool radius (1.7-9.5 nm) or [water]/[AOT] ratio ( w0) (8.0-43.0), the ΔpH and κ(i) increase maximally up to ∼-1.22 and ∼45, respectively, due to a greater water-exposed headgroup area. However, the unchanged ΔpH ≈ -0.65 and κ(i) ≈ 53.0 within radii ∼3.5-8.0 nm for the positive interface of a mixed Triton X-100 (TX-100)/SDS (4:1) micelle justify its packing flexibility. Interestingly, the continuously increasing ΔpH trend for IM up to its largest possible water-pool radius of ∼9.5 nm may rationalize the increase in ΔpH (∼-1.4 to -1.6) with the change in the curvature radii (∼15 to 50 nm) for sodium 1,2-dimyristoyl- sn-glycero-3-phosphorylglycerol (DMPG)/1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) (2:1) large unilamellar vesicles (LUV) owing to its negative interface. Whereas, similar to the micellar positive interface, the unchanged ΔpH at the positive LUV interface was confirmed by fluorescence microscopic studies with giant unilamellar vesicles of identical lipids composition. The present study offers a unique and simple method of monitoring the curvature-radius-dependent interfacial pH/polarity for biologically related membranes.

Influence of molecular shape on magnetic field effect on photo-induced geminate radical pair in SDS micellar medium

The magnetic field effects (MFEs) on the dynamics of the two radical pairs (RPs), one generated by photo-excitation of phenyl pyrilium ion (PP+) in the presence of SDS micelles and an electron donor, biphenyl, and the other similarly generated from a SDS micellar solution of biphenyl and trioxotriangulenium carbocation (OXO+), have been compared. At zero field, the RP (PP˙/BP˙+) has much higher recombination rate, but much lower escape rate in comparison to the RP (OXO˙ /BP˙+). The field-dependent yields and lifetimes show saturation early ( <0.1 T) in case of the latter, but in case of the former, the saturation does not occur, not even at a field of 5 T. The results have been interpreted in terms of relaxation mechanism (RM) for MFE and expected differences in the location of the guest in the micellar host, which affects the ratio of the rate of recombination to the rate of escape.