Insights into Protein Stability in Cell Lysate by 19F NMR Spectroscopy

In living organisms, protein folding and function take place in an inhomogeneous, highly crowded environment possessing a concentration of diverse macromolecules of up to 400 g/L. It has been shown that the intracellular environment has a pronounced effect on the stability, dynamics and function of the protein under study, and has for this reason to be considered. However, most protein studies neglect the presence of these macromolecules. Consequently, we probe here the overall thermodynamic stability of cold shock protein B from Bacillus subtilis (BsCspB) in cell lysate. We found that an increase in cell lysate concentration causes a monotonic increase in the thermodynamic stability of BsCspB. This result strongly underlines the importance of considering the biological environment when inherent protein parameters are quantitatively determined. Moreover, we demonstrate that targeted application of ¹⁹F NMR spectroscopy operates as an ideal tool for protein studies performed in complex cellular surroundings.     

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution ¹⁹F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.     

A Magic-Angle-Spinning NMR Spectroscopy Method for the Site-Specific Measurement of Proton Chemical-Shift Anisotropy in Biological and Organic Solids

Proton chemical shifts are a rich probe of structure and hydrogen-bonding environments in organic and biological molecules. Until recently, measurements of ¹H chemical-shift tensors have been restricted to either solid systems with sparse proton sites or were based on the indirect determination of anisotropic tensor components from cross-relaxation and liquid-crystal experiments. We have introduced a magic-angle-spinning approach that permits site-resolved determination of chemical-shift-anisotropy tensors of protons forming chemical bonds with labeled spin 1/2 nuclei in fully protonated solids with multiple sites, including organic molecules and proteins. This approach, originally introduced for the measurements of chemical-shift tensors of amide protons, is based on three RN-symmetry-based experiments, from which the principal components of the ¹H chemical-shift tensor can be reliably extracted by simultaneous triple fit of the data. Herein, we expand our approach to a much more challenging system involving aliphatic and aromatic protons. We start with a review of prior work on experimental NMR spectroscopy and computational quantum chemical approaches for measurements of ¹H chemical-shift tensors and relating these to the electronic structures. We then present our experimental results on U-¹³C,¹⁵N-labeled histidine and demonstrate that ¹H chemical-shift tensors can be reliably determined for the ¹H¹⁵N and ¹H¹³C spin pairs in cationic and neutral forms of histidine. Finally, we demonstrate that the experimental ¹H(C) and ¹H(N) chemical-shift tensors are in agreement with DFT calculations; therefore, establishing the usefulness of our method for the characterization of structures and hydrogen-bonding environments in organic and biological solids.     

Studies on the copolymerization of methyl methacrylate and N-aryl maleimides

This article describes the synthesis and characterization of copolymers of methyl methacrylate (MMA) and N-4-chlorophenyl maleimide (PC)/N-3-chlorophenyl maleimide (MC). The copolymers were synthesized by varying the mole fraction of N-aryl maleimides from 0.1 to 0.5 in the initial feed using azobisisobutyronitrile (AIBN) as an initiator and tetrahydrofuran (THF) as the solvent. The copolymer composition was determined from the ¹H-NMR spectra by taking the ratio of proton resonance signals due to methoxy protons (δ = 3.59 ppm) of MMA and aromatic protons (δ = 7.2–7.4 ppm) of N-aryl maleimides. The reactivity ratios for MMA–PC and MMA–MC copolymers were found to be 0.952 (r₁), 0.029 (r₂) and 0.833 (r₁) and 0.033 (r₂), respectively. Thermal characterization of the copolymers was done using differential scanning calorimetry (DSC) and dynamic thermo-gravimetry. Initial decomposition temperature and glass transition temperature increased with increasing mole fraction of N-aryl maleimide content in the copolymers. © 1996 John Wiley & Sons, Inc.     

Recent Developments on gMicroMC: Transport Simulations of Proton and Heavy Ions and Concurrent Transport of Radicals and DNA

Mechanistic Monte Carlo (MC) simulation of radiation interaction with water and DNA is important for the understanding of biological responses induced by ionizing radiation. In our previous work, we employed the Graphical Processing Unit (GPU)-based parallel computing technique to develop a novel, highly efficient, and open-source MC simulation tool, gMicroMC, for simulating electron-induced DNA damages. In this work, we reported two new developments in gMicroMC: the transport simulation of protons and heavy ions and the concurrent transport of radicals in the presence of DNA. We modeled these transports based on electromagnetic interactions between charged particles and water molecules and the chemical reactions between radicals and DNA molecules. Various physical properties, such as Linear Energy Transfer (LET) and particle range, from our simulation agreed with data published by NIST or simulation results from other CPU-based MC packages. The simulation results of DNA damage under the concurrent transport of radicals and DNA agreed with those from nBio-Topas simulation in a comprehensive testing case. GPU parallel computing enabled high computational efficiency. It took 41 s to simultaneously transport 100 protons with an initial kinetic energy of 10 MeV in water and 470 s to transport 105 radicals up to 1 µs in the presence of DNA.     

Cluster analysis of water molecules in alanine racemase and their putative structural role

Conservation of water molecules was identified by a cluster analysis of seven crystal structures of alanine racemase from Bacillus stearothermophilus. A total of 47 clusters of consensus water sites were determined and found to be highly localized, as indicated by their low mobilities. These clusters are located in the region of the active sites as well as at the interface between the N-terminal domain (the alpha/beta-barrel) of the first monomer and the C-terminal domain of the second monomer. The clusters located at the dimer interface form extensive hydrogen-bonding networks linked to the protein backbone. These water-mediated hydrogen bonds, and also all hydrogen-bonding interactions at the dimer interface, were monitored during a 2 ns molecular dynamics simulation and showed that when the inhibitor propionate was bound to the enzyme, some of these interactions were disrupted. The data we present here indicate that the consensus water sites identified at the interface between the two monomers of alanine racemase may play a structural role, which is to maintain and stabilize the alanine racemase dimer. A second role might be to supply the active site continuously with water molecules in order to allow rapid equilibration of active site protons with the solvent.     

Activity of Bromelain with Cationic Surfactants and the Correlation with the Change of 1H NMR Signals

This study observed the activities of bromelain in the presence of various cationic surfactants at different temperatures and the conformational changes in bromelain by ¹H NMR spectroscopy. We found that the bromelain activity was enhanced by tens to hundreds of micromoles per liter of the surfactant. In the presence of the surfactants, bromelain exhibited good tolerance to a range of substrate temperatures and its thermal stability was also increased. The ¹H NMR experiments indicated that when the temperature was increased from 25.0 to 45.0 °C, the protons of bromelain having chemical shifts (δ) between 3.7 and 5.2 ppm moved upfield, while those having δ values between 3.2 and 3.7 ppm moved downfield. In the bromelain/cationic surfactant mixture, the values of δ for the protons in both bromelain and the cationic surfactants decreased, accompanied by the broadening of the half-peak width of the surfactant protons. These results indicated that both increasing temperature and adding a cationic surfactant made the bromelain chain more flexible and hence, increased the bromelain activity. To the best of our knowledge, this was the first time that the relationship between the protein activity and the ¹H NMR data was expounded.     

Determination of positional distribution of butyryl groups in milkfat triacylglycerols,triacylglycerol mixtures,and isolated positional isomers of triacylglycerols by gas chromatography and1H nuclear magnetic resonance spectroscopy

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol;X,Y=long-chain acyls) of saturated triacylglycerols (TAG) with 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%) methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz¹H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The¹H NMR spectra of three interesterified mixtures of three monoacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups atsn-1(3)- andsn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.     

Solvent isotope effect on sol–gel transition of methylcellulose studied by DSC

Methylcellulose (MC), a hydrophobically modified cellulose derivative, in an aqueous solution undergoes sol-to-gel and gel-to-sol transitions on heating and cooling, respectively. Using differential scanning calorimetry, MC in light (H₂O) and heavy (D₂O) water solutions has been investigated to elucidate the solvent isotope effect on the transitions. As a result, their transition temperatures are higher in H₂O by about 4 °C than D₂O. This phenomenon is rationalized in terms of the strength of the hydrophobic attractive interaction; the strength is enhanced by D₂O. We discuss the reason for the enhancement and the difference in the isotope effect between MC and a poly(N-isopropylacrylamide) polymer which shows an opposite trend to MC.     

Thermoreversible gelation of a polystyrene–poly(ethylene/butylene)–polystyrene triblock copolymer in n‐octane/4‐methyl‐2‐pentanone solutions

The thermoreversible gelation of a triblock copolymer polystyrene‐block‐poly(ethylene/butylene)‐block‐polystyrene in n‐octane and two solvent mixtures of n‐octane and 4‐methyl‐2‐pentanone with a high n‐octane content has been studied. n‐Octane and 4‐methyl‐2‐pentanone are selective solvents for the middle poly(ethylene/butylene) block and the end polystyrene blocks, respectively. The influence of the solvent composition on the sol–gel transition and the mechanical properties of the gels was studied. The gel formation temperature increased with the copolymer concentration and the n‐octane content in the solvent system. The mechanical properties of the different gels were studied through oscillatory shear measurements. The concentration dependence of the elastic storage modulus showed an exponent close to that expected for gels in good solvents (2.25) that possess a structure similar to those of chemical networks. © 2002 Society of Chemical Industry     

The solubility of sulfur dioxide and hydrogen sulfide in associating solvents

The solubilities of SO₂ are reported in the solvents N, N-dimethyl acetamide (DMA), N, N-dimethyl formamide (DMF), ethyl acetate, acetonitrile, chlorobenzene, methanol, (1,2-ethanediol) ethylene glycol and acetone for atmospheric pressure and for temperatures ranging from 268 K to 333 K (-5°C to 60°C). Solubilities of H₂S are also reported for the first five of the above-mentioned solvents and for hexane for similar conditions. These gases and solvents exhibit extreme molecular interactions which are reflected in unusual solubility behavior. This paper is a continuing attempt in providing data and analyses for improving the understanding of gas solubilities in complex gas-solvent systems. Solubilities expressed as hydrogen-bonding factors have been found useful in systematically relating gas solubilities in one associating or reacting solvent to another chemically similar solvent. Thus, with a limited amount of data for gas solubilities, it is often possible to predict solubilities even in highly associated or reacting gas-solvent solutions.     

Quantitative structure–activity relationships (QSARs) as a tool for predicting the sorption of organic chemicals in soils

The use of quantitative structure-activity relationships (QSARs) based on linear free-energy relationships (LFERs), molecular connectivity (MC) indices, and on more sophisticated quantum mechanical approaches, for predicting soil sorption coefficients is reviewed. The nature and energetics of sorption of organic chemicals by soils are also briefly reviewed. On the basis of the data presented we may conclude that from the large body of physicochemical parameters, the n-octanol/water partition coefficient (K_ow) has been most successful in modeling sorption data of organic chemicals. From the large set of nonempirical parameters that are defined in terms of a compound's 2- or 3-dimensional structure and can be derived for any chemical without experimental efforts, the MC indices have proven the most reliable and easily available.     

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

We have investigated 4‐halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4‐chloropyridine with amino acids and thiols was ranked with respect to common covalent protein‐modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment‐sized 4‐halopyridines inactivated human dimethylarginine dimethylaminohydrolase‐1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off‐pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2‐fluoromethyl‐substituted 4‐chloropyridines demonstrated that the pK_a and k_inact/K_I values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4‐Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.     

Thermodynamic Stability of Low‐k Amorphous SiOCH Dielectric Films

Si–O–C‐based amorphous or nanostructured materials are now relatively common and of interest for numerous electronic, optical, thermal, mechanical, nuclear, and biomedical applications. Using plasma‐enhanced chemical vapor deposition (PECVD), hydrogen atoms are incorporated into the system to form SiOCH dielectric films with very low dielectric constants (k). While these low‐k dielectrics exhibit chemical stability as deposited, they tend to lose hydrogen and carbon (as labile organic groups) and convert to SiO₂ during thermal annealing and other fabrication processes. Therefore, knowledge of their thermodynamic properties is essential for understanding the conditions under which they can be stable. High‐temperature oxidative drop solution calorimetry measurement in molten sodium molybdate solvent at 800°C showed that these materials possess negative formation enthalpies from their crystalline constituents (SiC, SiO₂, C, Si) and H₂. The formation enthalpies at room temperature become less exothermic with increasing carbon content and more exothermic with increasing hydrogen content. Fourier transform infrared spectroscopy (FTIR) spectroscopy examined the structure from a microscopic perspective. Different from polymer‐derived ceramics with similar composition, these low‐k dielectrics are mainly comprised of Si–O(C)–Si networks, and the primary configuration of carbon is methyl groups. The thermodynamic data, together with the structural analysis suggest that the conversion of sp² carbon in the matrix to surface organic functional groups by incorporating hydrogen increases thermodynamic stability. However, the energetic stabilization by hydrogen incorporation is not enough to offset the large entropy gain upon hydrogen release, so hydrogen loss during processing at higher temperatures must be managed by kinetic rather than thermodynamic strategies.     

NMR characterization of dihydrosterculic acid and its methyl ester

Cyclopropane FA occur in nature in the phosphoplipids of bacterial membranes, in oils containing cyclopropene FA, and in Litchi sinensis oil. Dihydrosterculic acid (2-octyl cyclopropaneoctanoic acid) and its methyl ester were selected for ¹H and ¹³C NMR analysis as compounds representative of cyclopropane FA. The 500 MHz ¹H NMR spectra acquired with CDCl₃ as solvent show two individual peaks at −0.30 and 0.60 ppm for the methylene protons of the cyclopropane ring. Assignments were made with the aid of 2D correlations. In accordance with previous literature, the upfield signal is assigned to the cis proton and the downfield signal to the trans proton. This signal of the trans proton is resolved from the peak of the two methine protons of the cyclopropane ring, which is located at 0.68 ppm. The four protons attached to the two methylene carbons α to the cyclopropane ring also show a split signal. Two of these protons, one from each methylene moiety, display a distinct shift at 1.17 ppm, and the signal of the other two protons is observed at 1.40 ppm, within the broad methylene peak. The characteristic peaks in the ¹³C spectra are also assigned.     

Coupled Transport of Zr(IV) through Tri-n-butylphosphate-Xylene-Based Supported Liquid Membranes

Abstract

The transport of Zr(IV) through tri-n-butylphosphate-xylene-based liquid membranes, supported in a polypropylene hydrophobic microporous film, has been studied. The concentration of HNO₃ in the feed solution and tri-n-butylphosphate (TBP) carrier in the membrane were varied, and the flux and permeability coefficients were determined. The optimum conditions found for maximum flux were determined to be 10 mol/dm³ HNO₃ and 2.93 mol/dm³ TBP with a flux value of 12.9 × 10^?6 mol · m^?2 · s^?1. The solvent extraction study revealed that 1.25 to 3.5 protons are involved in zirconium transport, and that two molecules of TBP are involved in the complex formation. The value of protons involved varies with acid concentration. The zirconium ion transport is coupled with nitrate ions transport.     

Semi- and fully interpenetrating polymer networks based on polyurethane–polyacrylate systems. I. The polyurethane networks

The polyurethane networks based on commerical prepolymer, Adiprene L-100, and trimethylol propane (system 1) and on toluene diisocyanate, polypropylene gylcol, and trimethylol propane (system 2) were prepared and characterized in a number of ways. The materials constitute the first formed networks in a series of interpenetrating polymer networks and semi-interpenetrating polymer networks to be reported in subsequent papers in this series. System 1 networks were characterized by swelling tests which showed the M _c values to be sensitive to the amount of polyurethane present in the polymerization solvent. Stress–strain, stress–relaxation, and dynamic mechanical analyses wer also conducted. For system 2, M _c was measured, by both the swelling and the Mooney–Rivlin techniques, for materials in which the diol-to-triol ratios had been altered. the latter showed C₁ increasing as M _c decreased while C₂ was small and changed onlyy slightly indicating approximately ideal behavior. These M _c values were about 13 % larger than predicted by swelling.     

Lösungsmittelinduzierte Elektronenstrukturänderungen bei negativ solvatochromen Farbstoffen

Solvent-induced Changes of the Electronic Structure of Negatively Solvatochromic Dyes The ¹H NMR chemical shifts of negatively solvatochromic dyes depend on the polarity of the solvent used. The differences of the chemical shifts of vicinal protons decrease with increasing polarity. This result supports the idea that the electronic structure of negatively solvatochromic dyes changes from a polymethine-like state in non-polar solvents to a polyene-like state in polar solvents.     

B-factor Guided Proline Substitutions in Chromobacterium violaceum Amine Transaminase: Evaluation of the Proline Rule as a Method for Enzyme Stabilization

Biocatalysis is attracting interest in the chemical industry as a sustainable alternative in large-scale chemical transformations. However, low operational stability of naturally evolved enzymes is a challenge and major efforts are required to engineer protein stability, usually by directed evolution. The development of methods for protein stabilization based on rational design is of great interest, as it would minimize the efforts needed to generate stable enzymes. Here we present a rational design strategy based on proline substitutions in flexible areas of the protein identified by analyzing B-factors. Several proline substitutions in the amine transaminase from Chromobacterium violaceum were shown to have a positive impact on stability with increased half-life at 60 °C by a factor of 2.7 (variant K69P/D218P/K304P/R432P) as well as increased melting temperature by 8.3 °C (variant K167P). Finally, the presented method utilizing B-factor analysis in combination with the proline rule was deemed successful at increasing the stability of this enzyme.     

Deactivation of 6-Aminocoumarin Intramolecular Charge Transfer Excited State through Hydrogen Bonding

This paper presents results of the spectral (absorption and emission) and photophysical study of 6-aminocoumarin (6AC) in various aprotic hydrogen-bond forming solvents. It was established that solvent polarity as well as hydrogen-bonding ability influence solute properties. The hydrogen-bonding interactions between S₁-electronic excited solute and solvent molecules were found to facilitate the nonradiative deactivation processes. The energy-gap dependence on radiationless deactivation in aprotic solvents was found to be similar to that in protic solvents.