Solvation dynamics of a protein in the pre molten globule state

The nature of solvent molecules around proteins in native and different non-native states is crucial for understanding the protein folding problem. We have characterized two compact denatured states of glutaminyl-tRNA synthetase (GlnRS) under equilibrium conditions in the presence of a naturally occurring osmolyte, l-glutamate. The solvation dynamics of the compact denatured states and the fully unfolded state has been studied using a covalently attached probe, acrylodan, near the active site. The solvation dynamics progressively becomes faster as the protein goes from the native to the molten globule to the pre molten globule to the fully unfolded state. Anisotropy decay measurements suggest that the pre-molten-globule intermediate is more flexible than the molten globule although the secondary structure is largely similar. Dynamic light scattering studies reveal that both the compact denatured states are aggregated under the measurement conditions. The implications of solvation dynamics in aggregated compact denatured states have been discussed.     

2,2,2-trifluoroethanol-induced molten globule state of concanavalin a and energetics of 8-anilinonaphthalene sulfonate binding: calorimetric and spectroscopic investigation

The interaction of 2,2,2-trifluoroethanol (TFE) with concanavalin A has been investigated by using a combination of differential scanning calorimetry, isothermal titration calorimetry (ITC), circular dichroism (CD), and fluorescence spectroscopy at pH 2.5 and 5.2. All of the calorimetric transitions at both the pH values were found to be irreversible. In the presence of 4 mol kg(-1) TFE at pH 2.5, concanavalin A is observed to be in a partially folded state with significant loss of native tertiary structure. The loss of specific side chain interactions in the transition from native to the TFE-induced partially folded state is demonstrated by the loss of cooperative thermal transition and reduction of the CD bands in the aromatic region. Acrylamide quenching, 8-anilinonaphthalene sulfonate (ANS) binding, and energy transfer also suggest that in the presence of 4 mol kg(-1) TFE at pH 2.5 concanavalin A is in a molten globule state. ITC has been used for the first time to characterize the energetics of ANS binding to the molten globule state. ITC results indicate that the binding of ANS to the molten globule state and acid-induced state at pH 2.5 displays heterogeneity with two classes of non-interacting binding sites. The results provide insights into the role of hydrophobic and electrostatic interactions in the binding of ANS to concanavalin A. The results also demonstrate that ITC can be used to characterize the partially folded states of the protein both qualitatively and quantitatively.     

Differential scanning calorimetric study of the molten globule state of cytochrome c induced by sodium n-dodecyl sulfate

The molten globule (MG) state, a compact denatured state with a significantly native-like secondary structure but a largely flexible and disordered tertiary structure, has been proposed to be a major intermediate of protein folding. To explore another approach for characterizing the MG state, sodium dodecyl sulfate (SDS) induced formation of the MG state of horse cytochrome c at pH 2 was studied by circular dichroism, visible spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). These techniques confirmed that the addition of SDS to acid-unfolded state of cytochrome c induced the MG state. Although, the DSC thermal denaturation of cytochrome c was always calorimetrically irreversible, the MG state induced by SDS at low concentrations showed a reversible profile. The spectroscopic properties demonstrated that the hydrophobic tail of SDS utilized the hydrophobic contribution to stabilizing the heme conformation at MG state in cytochrome c. This would be the main reason of thermal profile reversibility of MG state in cytochrome c. The reversibility of DSC thermogram would allow its deconvolution and analysis of the energetic domains for this protein.     

Mechanism for stabilization of the molten globule state of papain by sodium n-alkyl sulfates: spectroscopic and calorimetric approaches

Papain exists in a molten globule (MG) state at pH 2 and in this state protein tends to aggregate in the presence of lower concentrations of guanidine hydrochloride (GuHCl). Such aggregation is prevented if low concentrations of sodium n-alkyl sulfates are also present in the buffer; in addition, stabilization of the protein is also induced. The guanidine hydrochloride and temperature-induced unfolding of papain, in the presence of n-alkyl sulfates, indicate stabilization of the protein as seen from the higher transition midpoints when monitored by fluorescence, circular dichroism, and differential scanning calorimetry. However, a similar phenomenon is not seen under neutral conditions in the presence of n-alkyl sulfate concentrations. The effect of n-alkyl sulfates on the structure of the MG state of papain was utilized to investigate the contribution of hydrophobic interaction to the stability of the MG state. The Td values of the MG states of papain in the presence of n-alkyl sulfates at different concentrations showed substantial variation. The enhancement of Td values at the stability criterion of MG states corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the hydrophobic interactions play important roles in stabilizing and preventing the aggregation of the MG state of papain.     

Origin of miscibility‐induced sequential reordering and crystallization‐induced sequential reordering in binary copolyesters: a Monte Carlo simulation

The effect of the repulsive interaction between the components of binary copolyesters on their sequence order was investigated with the Monte Carlo simulation method. The phase separation and ester‐interchange reactions were implemented simultaneously with a kind of one‐site bond fluctuation model. When the repulsive interaction energy was applied to the binary copolyesters, miscibility‐induced sequential reordering (MISR) was induced. The more repulsive the pair interaction was, the higher the sequence order was. During the MISR process, homoester‐interchange reactions became more favorable because of the repulsive interaction, accompanying the decrease of the interactional free energy. The sequence order resulting from MISR was independent of the relative trial ratio of phase separation to ester‐interchange reaction at a given value of interaction energy. Restoration of the sequence distribution was also simulated with and without the repulsive interaction between the components of the binary copolyesters to investigate the effect of MISR on the crystallization‐induced sequential reordering (CISR) process in binary copolyesters, where sequences with lengths longer than 6 were assumed to crystallize and could not take part in ester‐interchange reactions. The sequence distribution in the amorphous phase was restored via ester‐interchange reactions. When the repulsive interaction was applied to binary copolyesters during the CISR process, restoration of the sequence distribution was accelerated, indicating that MISR can accelerate the CISR process when a polyester blend shows upper critical solution temperature behavior. © 2001 John Wiley & Sons, Inc. J Polym Sci Part B: Polym Phys 39: 1337–1347, 2001     

Determination of the thiol redox state of organisms: new oxidative stress indicators

This study describes a new methodology by which the concentrations of non-protein (NP) thiols glutathione (GSH), cysteine (CSH), N-acetylcysteine (AcCSH), and protein (P) thiols (PSH), as well as the contribution of these components to symmetric and mixed disulfides (NPSSR, NPSSC, NPSSCAc, PSSR, PSSC, PSSCAc, PSSP) can reliably be measured. The methodology consists of a strict sequence of methods which are applied to every sample. Free thiols at any given state of the procedure are measured by Ellmans assay, the CSH fraction is measured by its unique response in the ninhydrin assay, AcCSH is selectively measured with ninhydrin after enzymatic deacylation, proteins are separated from non-protein thiols/disulfides by precipitation with trichloroacetic or perchloric acid, disulfides are reduced into free thiols with borohydride, mixed disulfides between a protein and a non-protein component are measured by extracting the non-protein thiol from the protein pellet after borohydride treatment, and protein thiols/disulfides are measured after resolubilization of the protein pellet.When this method was applied to animal and fungal tissue, new molecular indicators of the thiol redox state of living cells were identified. The findings of the present study clearly show that the new parameters are very sensitive indicators of redox state, while at the same time the traditional parameters GSH and GSSG often remain constant even upon dramatic changes in the overall redox state of biological tissue. Therefore, unbiased assessment of the redox state also requires explicit measurement of its most sensitive thiol indicators.Electronic Supplementary Material Supplementary material is available in the online version of this article at . A link in the frame on the left on that page takes you directly to the supplementary material.     

Drug design with a new transition state analog of the hydrated carbonyl: silicon-based inhibitors of the HIV protease

BACKGROUND: Silicon is the element most similar to carbon, and bioactive organosilanes have therefore been of longstanding interest. Design of bioactive organosilanes has often involved a systematic replacement of a bioactive molecule's stable carbon atoms with silicon. Silanediols, which are best known as unstable precursors of the robust and ubiquitous silicone polymers, have the potential to mimic an unstable carbon, the hydrated carbonyl. As a bioisostere of the tetrahedral intermediate of amide hydrolysis, a silanediol could act as a transition state analog inhibitor of protease enzymes. RESULTS: Silanediol analogs of a carbinol-based inhibitor of the HIV protease were prepared as single enantiomers, with up to six stereogenic centers. As inhibitors of this aspartic protease, the silanediols were nearly equivalent to both their carbinol analogs and indinavir, a current treatment for AIDS, with low nanomolar K(i) values. IC(90) data from a cell culture assay mirrored the K(i) data, demonstrating that the silanediols can also cross cell membranes and deliver their antiviral effects. CONCLUSIONS: In their first evaluation as inhibitors of an aspartic protease, silanediol peptidomimetics have been found to be nearly as potent as currently available pharmaceutical agents, in enzyme and cell protection assays. These neutral, cell-permeable transition state analogs therefore provide a novel foundation for the design of therapeutic agents.     

Synthesis and fluorescent properties of a new photostable thiol reagent “BACM”

The 3-maleimidocoumarin BACM ( 2d ) is a new tag designed to react with thiols in biological materials to form fluorescent derivatives whose emission is in the 500 nm (green) region with good resistance to fading. The fluorescence properties of its N-acetyl-L-cysteine derivative are described. BACM itself is non-fluorescent, as is its predecessor, DACM ( 1a ). A synthesis of BACM is reported which avoids the formation of nitrocoumarin isomers obtained in the reported synthesis of DACM. The synthesis of the related 3-(4-maleimidophenyl)coumarin is also described.     

Evidence for unfolding and refolding of gas-phase cytochrome <Emphasis Type="Italic">c</Emphasis> ions in a Paul trap

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.     

Synthesis and adsorption on gold surfaces of a functionalized thiol: elaboration and test of a new nitroaromatic gas sensor

A new type of sensor was built by synthesizing a long-chain thiol functionalized with an aromatic head group and grafting it onto a gold surface. The synthesis route is here described, together with the IR, MS, and RMN analysis of the new product. Adsorption of the latter onto gold was assessed by a combination of RAIRS and XPS data. Those reveal that a monolayer of thiol is adsorbed and oriented with the benzene groups toward the external part of the layer. Detection tests were performed in various atmospheres by QCM. The response shows good sensitivity to 2,4-dinitrotrifluoromethoxybenzene as a model of nitroaromatic compound.     

Übergangsmetallchalkogenverbindungen. Evidence for the existence of new Thioanions of Vanadium and Rhenium by their electronic spectra

The reaction of MO (M = V, Re) with H₂S investigated by spectrophotometry, indicated the existence of two new thioanions VO₂S and ReOS. Their spectra are compared with known relationships in those of the thioanions of molybdenum and tungsten. A proposal for the electronic spectrum of VO₃S^3? is given.     

Structural and electronic properties of new fullerene derivatives and their possible application as HIV-1 protease inhibitors

Density functional theory (DFT) calculations have been carried out at the hybrid Becke 3-Lee–Yang–Parr; B3LYP/3-21G** level of theory to study two series of hydroxy-chalca-acetic acid-(4-pyrrolidin-1-yl-phenyl) ester [C₆₀-C₂H₄N-(4-XCOCH₂OH)C₆H₄] and hydroxy-chalcoacetic acid-[2-(2-hydroxy-acetylchalcanyl)-4-pyrrolidin-1-yl-phenyl] ester[C₆₀-C₂H₄N-(3,4-XCOCH₂OH)C₆H₄]. The X atom is O, S or Se for the two series. The vibrational spectra, physical, chemical, thermodynamics and Quantitative Structure Activity Relationship (QSAR) properties of the studied molecules are calculated and discussed. We have evaluated these molecules as HIV-1 protease inhibitors based on the hydrogenation interaction between the hydroxymethylcarbonyl (HMC) groups and the two aspartic acid of the HIV-1 protease active site. Results show that some of the investigated fullerene-based derivatives can be considered promising as HIV-1 protease inhibitors.     

Optical spectroscopic and TEM studies of catanionic micelles of CTAB/SDS and their interaction with a NSAID

If a vesicle is a better model of a membrane in the context of the hydrophobic effect, then from the charge distribution point of view, a catanionic micelle is a closer model to a biomembrane. We have prepared and characterized two different types of catanionic micelles of sodium dodecyl sulfate (SDS) and cetyl N,N,N-trimethylammonium bromide (CTAB) having different surface charge ratios using optical spectroscopy and transmission electron microscopy. The average size of both types of mixed micelles was found to be much larger than that of micelles containing uniformly charged headgroups. Catanionic micelles containing higher concentrations of positively charged headgroups (CTAB) are larger in size, less compact, and more polar compared to the micelles containing higher concentrations of negatively charged headgroups (SDS). We have used these catanionic micelles as membrane mimetic systems to understand the interaction of piroxicam, a nonsteroidal anti-inflammatory drug (NSAID) of the oxicam group, with biomembranes. In continuation of our work on membrane mimetic systems, we have used spectral properties of the drug itself to understand the effect of the presence of mixed charges on the micellar surface in guiding the interaction of catanionic micelles with piroxicam. Our earlier studies of the interaction of piroxicam with micelles having uniform surface charges have shown that the charge on the micellar surface not only dictates which prototropic form of the drug will be incorporated in the micelles but also induces a switch-over between different prototropic forms of piroxicam. The equilibrium of this switch-over is extremely sensitive to the environment. In this study, we demonstrate how even small changes in the electrostatic forces obtained by doping the uniformly charged surface of the micelles with oppositely charged headgroups (as in catanionic micelles) are capable of fine-tuning this equilibrium. This implies that the surface charge of biomembranes, which are quite diverse in vivo, might play a significant role in selecting a particular form of the drug to be presented to its targets.     

The densities and molar volumes of molten lead(II) dodecanoate/dodecanoic acid mixtures over their complete composition range: Evidence for non-ideal behaviour

Data are presented for the densities and molar volumes of the molten system lead(II) dodecanoate/dodecanoic acid over its complete composition range. For equimolar mixtures, plots of molar volume against temperature show curvature at high temperatures, suggesting deviations from ideal behaviour. Support for this comes from a plot of molar volume at constant temperature against acid mole fraction. Densities and molar volumes are reported for lead (II) carboxylate/carboxylic acid (0.5 mole fraction) and for pure carboxylic acid for the even chain acids C₁₀ to C₁₈. The molar volumes at constant temperature in these cases are linear functions of chain length, although the volume occupied per methylene group in equimolar mixtures is suggested to be slightly smaller than with pure soap or pure acid. An explanation for non-ideal behaviour becoming more marked at higher temperature is given in terms of acid monomer-dimer equilibria.     

Photodissociation of Co3: Evidence for a long-lived excited state

Energy analysis of the photofragment O^? ions produced in the photodissociation reaction CO₃^? + hv »- O^? + CO₂ identifies two dinstict O^? production mechanisms: a two-photon absorption via an intermediate bound electronic state, and a collision-assisted single-photon process via a long-lived excited state. This species has a radiative lifetime exceeding one microsecond. and a collisional dissociation cross section measurably higher than that of the ground state.     

Thermal stability of P3HT and P3HT:PCBM blends in the molten state

The thermal stability of poly(3-hexylthiophene) (P3HT) in its molten state was investigated in air and nitrogen atmospheres under no illumination conditions, with the aim of testing the feasibility of processing it using polymer melt techniques. A large set of different experimental characterization techniques was used including thermogravimetric analysis (TGA), rotational rheometry, infrared spectroscopy (FTIR-ATR), proton nuclear magnetic resonance spectroscopy (¹H-NMR), gel permeation chromatography (GPC), UV-Vis and fluorescence spectroscopy. The results obtained strongly suggest that the processing of P3HT in its molten state is possible, without noticeable degradation, if carried out under nitrogen atmosphere and if the processing (residence) times are relatively short. Conversely, as expected, in a normal air atmosphere P3HT degrades rapidly at temperatures above its melting point. The effect of PCBM on the thermal stability of P3HT:PCBM blends in the molten state was also studied using TGA, and in air atmosphere PCBM is shown to delay oxidation.     

Density functional evaluation and a feasibility study of intramolecular thione–thiol tautomerization

Performance of various density functionals for use in geometry and energetics calculation of thiocarbonyl systems is evaluated. The three biologically significant systems such as 2‐methyl‐3‐thiosemicarbazide, 1‐acetyl‐2‐thiohydantoin and bornane‐2‐thione are selected for the geometry analysis. Energy of tautomerization of thioacetone and enthalpy of formation of 2‐thiohydantoin are considered for analyzing the performance in energetics. Minnesota functionals such as M05‐2X, M06‐2X and range separated hybrid functional wB97 give best results in both geometry and energetics calculations. Studies on basis set effect showed reliable performance by 6‐31 + G* and 6‐31++G** over higher basis sets. Feasibility of tautomerization in the aforementioned thiones has been investigated using the best performing density functionals. The proposed thione–thiol tautomerization step during PPC‐DC catalyzed decarboxylation reaction has also been investigated and the computed high energy barrier shows less feasibility of the reaction.