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PubMed2005

Helix stability and hydrophobicity in the folding mechanism of the bacterial immunity protein Im9.

Recent models suggest that the mechanism of protein folding is determined by the balance between the stability of secondary structural elements and the hydrophobicity of the sequence. Here we determine the role of these factors in the folding kinetics of Im9* by altering the secondary structure propensity or hydrophobicity of helices I, II or IV by the substitution of residues at solvent exposed sites. The folding kinetics of each variant were measured at pH 7.0 and 10 degrees C, under which conditions wild-type Im9* folds with two-state kinetics. We show that increasing the helicity of these sequences in regions known to be structured in the folding intermediate of Im7*, switches the folding of Im9* from a two- to three-state mechanism. By contrast, increasing the hydrophobicity of helices I or IV has no effect on the kinetic folding mechanism. Interestingly, however, increasing the hydrophobicity of solvent-exposed residues in helix II stabilizes the folding intermediate and the rate-limiting transition state, consistent with the view that this helix makes significant non-native interactions during folding. The results highlight the generic importance of intermediates in folding and show that such species can be populated by increasing helical propensity or by stabilizing inter-helix contacts through non-native interactions.

Bacterial Proteins↗
PubMed2000

Gutta-percha softening: "Hemo-De" as a xylene substitute.

The putative hazardous nature of chloroform and xylene implies that safer substitutes should be considered. Standard cylindrical gutta-percha samples were immersed in Hemo-De, a xylene substitute, for 60 s at 37 degrees C. Weight loss of a sample, after drying, was used as a measure of its solubility. Master and accessory cone gutta-percha of three brands were compared. The highest solubility of all kinds of gutta-percha was in chloroform, which served as a positive control. The average solubility of all samples in xylene and Hemo-De was 61% and 52% of that in chloroform, respectively. DMS gutta-percha was more soluble than of Hygienic and DeTrey. Master cone gutta-percha of all brands was more soluble than that of their accessory cones. These results indicate that (a) large differences exist in the solubility of gutta-percha and (b) Hemo-De dissolved gutta-percha in a range similar to that of xylene and may be considered as a potential substitute for this organic solvent.

Chloroform↗
PubMed1990

Tertiary structural constraints on protein evolutionary diversity: templates, key residues and structure prediction.

The pattern of residue substitution in divergently evolving families of globular proteins is highly variable. At each position in a fold there are constraints on the identities of amino acids from both the three-dimensional structure and the function of the protein. To characterize and quantify the structural constraints, we have made a comparative analysis of families of homologous globular proteins. Residues are classified according to amino acid type, secondary structure, accessibility of the sidechain, and existence of hydrogen bonds from sidechain to other sidechains or peptide carbonyl or amide functions. There are distinct patterns of substitution especially where residues are both solvent inaccessible and hydrogen bonded through their sidechains. The patterns of residue substitution can be used to construct templates or to identify 'key' residues if one or more structures are known. Conversely, analysis of conversation and substitution across a large family of aligned sequences in terms of substitution profiles can allow prediction of tertiary environment or indicate a functional role. Similar analyses can be used to test the validity of putative structures if several homologous sequences are available.

Amino Acid Sequence↗
PubMed1997

Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans.

The gene chiA, which codes for endochitinase, was cloned from a soilborne Enterobacter agglomerans. Its complete sequence was determined, and the deduced amino acid sequence of the enzyme designated Chia_Entag yielded an open reading frame coding for 562 amino acids of a 61-kDa precursor protein with a putative leader peptide at its N terminus. The nucleotide and polypeptide sequences of Chia_Entag showed 86.8 and 87.7% identity with the corresponding gene and enzyme, Chia_Serma, of Serratia marcescens, respectively. Homology modeling of Chia_Entag's three-dimensional structure demonstrated that most amino acid substitutions are at solvent-accessible sites. Escherichia coli JM109 carrying the E. agglomerans chiA gene produced and secreted Chia_Entag. The antifungal activity of the secreted endochitinase was demonstrated in vitro by inhibition of Fusarium oxysporum spore germination. The transformed strain inhibited Rhizoctonia solani growth on plates and the root rot disease caused by this fungus in cotton seedlings under greenhouse conditions.

Amino Acid Sequence↗
PubMedDate not supplied

[Trichloroethylene poisoning. Presentation of 3 cases and review of the literature].

Three cases of occupational poisoning by trichloroethylene, one of them fatal, are presented. The etiologic, clinical and diagnostic aspects of each case are discussed, along with a broad literature review, emphasizing preventive measures and the substitution of this chloric solvent by other, less toxic solvents, as recommended by the U.S. Food and Drug Administration.

Adult↗
PubMed1999

Effect of solvent on the characteristics of pentamidine loaded microcapsule.

Biodegradable microcapsules of pentamidine/poly(L-lactide-co-D,L-lactide) were prepared by solvent evaporation technique using a mixture of organic solvents. The control batch of microcapsules was prepared using dichloromethane. The effect of solvent on the characteristics of pentamidine loaded microcapsules was examined by substituting up to 30% of dichloromethane with acetone, methanol, DMSO, ethyl acetate, and ethanol, respectively. No significant change in the surface morphology was observed when dichloromethane was substituted with 20% or less amount of other solvents. These microcapsules were all porous and spherical. However, the use of 30% DMSO or ethanol, along with dichloromethane, resulted in a mixture of elongated and spherical microcapsules. The efficiency of encapsulation of these two batches was also significantly higher than the other batches of microcapsules. The average particle size of the microcapsules prepared with 30% DMSO (165 microm) was significantly higher than the other batches (< 80 microm). A substitution of 10-30% dichloromethane with other listed organic solvents also showed a significant difference in the initial drug release. The drug release within the first twenty-four hours varied from 4 to 16%. The use of a second organic solvent, except ethanol, resulted in a significantly higher drug release during the second half of the dissolution study. The drug release continued more than 60 days.

Acetates↗
PubMed1992

Vegetable oils instead of xylene in tissue processing.

In an effort to improve working conditions in the histopathologic laboratory, we have investigated whether less toxic substances may be substituted for the toxic organic solvent xylene currently in use. Xylene is used in the clearing as well as the deparaffinization process. We have tested whether the substitution of xylene by olive oil and coconut oil leads to any difference in the quality of the histologic sections. Two tissue blocks from each of 232 specimens sent for histopathologic evaluation were subjected to parallel processing in xylene and oil. The specimens represented a broad spectrum of tissue types. All sections were stained with hematoxylin-eosin to permit evaluation of histologic and cytologic details. Furthermore, a range of histochemical and immunohistochemical stainings were applied to a subgroup of tissue sections. The results showed qualitative differences between xylene-processed and oil-processed tissue in only a minority of cases. In no cases were the oil-processed samples considered less suitable for histologic diagnosis. In the histochemical and immunohistochemical stainings, no differences were registered. Although the long-term stability of oil-processed tissue remains to be clarified, we conclude that less toxic vegetable oil may probably be substituted for xylene without losing valuable diagnostic information.

Histological Techniques↗
PubMed2005

Theoretical studies of the tautomeric equilibria for five-member N-heterocycles in the gas phase and in solution.

Tautomeric equilibria have been studied for five-member N-heterocycles and their methyl derivatives in the gas phase and in different solvents with dielectric constants of epsilon = 4.7-78.4. The free energy changes differently for tautomers upon solvation as compared to the gas phase, resulting in a shift of the equilibrium constant in solution. Solvents with increasing dielectric constant produce more negative solute-solvent interaction energies and increasing internal energies. The methyl-substituted imidazole and pyrrazole form delicate equilibria between two tautomeric forms. Depending on the solvent, the methyl-substituted triazoles and tetrazole have one or two major tautomers in solution. When estimating the relative solvation free energies by means of an explicit solvent model and using the FEP/MC method, one observes that the preferred tautomers differ in several cases from those predicted by the continuum solvent model. The 1,2-prototropic shift, as an intramolecular tautomerization path, requires about 50 kcal/mol activation energy for imidazole in the gas phase, and this route is also disfavored in a solution. The calculated activation free energy along the intramolecular path is 48-50 kcal/mol in chloroform and water as compared to a literature value of 13.6 kcal/mol for pyrrazole in DMSO. A molecular dynamics computer experiment favors the formation of an imidazole chain in chloroform, making the 1,3-tautomerization feasible along an intermolecular path in nonprotic solvents. In aqueous solution, one strong N-H...Ow hydrogen bond is formed for each species, whereas all other nitrogens in the ring form weaker, N...HwOw type hydrogen bonds. The tetrahydrofuran solvent acts as a hydrogen bond acceptor and forms N-H... Oether bonds. Molecules of the dichloromethane solvent are in favorable dipole-dipole interactions with the solute. The results obtained are useful in the design of N-heterocyclic ligands forming specified hydrogen bonds with protein side chains.

Gases↗
PubMed1991

Animal experiments on the allergenicity of d-limonene--the citrus solvent.

With the increasing use of d-limonene as a substitute for chlorinated hydrocarbons, chlorofluorocarbons (CFC) and other organic solvents, a demand has arisen for more knowledge of the health effects of this substance. The aim of the present study was to investigate the allergenic effect of d-limonene on skin. The sensitizing potential of d-limonene and the influence of air exposure on its allergenicity were studied in guineapigs. d-Limonene of high purity gave no significant allergic reactions, while d-limonene exposed to air for 2 months sensitized the animals. Gas chromatographic analyses indicated that the content of limonene oxide in limonene increased with prolonged air exposure. It is concluded that allergenic compounds are formed from d-limonene upon prolonged air exposure. Products containing d-limonene should therefore be kept in cold storage in closed vessels. Manual handling should be avoided, or gloves worn, in order to reduce the risk of sensitization.

Animals↗
PubMed1992

Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability.

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.

Alanine↗
PubMed1998

Phosphorescence emission of 7-azatryptophan and 5-hydroxytryptophan in fluid solutions and in alpha2 RNA polymerase.

The tryptophan analogues 7-azaindole (7-Aza W) and 5-hydroxytryptophan (5-OH W) have a significant absorbance between 310-320 nm, which allows them to act as selective luminescence probes in protein mixtures containing a large number of tryptophan residues. To assess the potential of their phosphorescence emission in probing the nature of protein environments the delayed emission was examined as a function of temperature and solvent viscosity. Whereas in low temperature (135 K) propylene glycol/buffer glasses the phosphorescence of both 7-aza W and 5-OH W is structured, intense and exhibit a lifetime of a few seconds, above the glass transition temperature (180 K) the delayed emission is considerably quenched. Temperature profiles show that as the solvent is more fluid the phosphorescence of 5-OH W becomes red shifted, poorly structured and the triplet lifetime drops steeply reaching 29 micro(s) in buffer at 274 K. For 7-aza W the alterations are more drastic and no phosphorescence could be detected above 193 K. This implies that in fluid aqueous media the excited triplet state of these analogues is deactivated by vary efficient nonradiative processes. The quenching of 5-OH phosphorescence is not prevented even when the chromophore is inserted in a solvent protected protein environment. Indeed, substitution of the single Trp of a2 RNA Polymerase, which according to its relatively low phosphorescence lifetime at ambient temperature is substantially shielded from the solvent, with 5-OH did not inhibit the quenchability of the latter. Knowledge of the quenching mechanisms is therefore needed for this emission to report on the nature of the protein environment.

5-Hydroxytryptophan↗
PubMed2002

Novel synthesis of 2-chloroquinolines from 2-vinylanilines in nitrile solvent.

2-Vinyl- or heteroaryl-substituted anilines were reacted with diphosgene in acetonitrile solution via a reactive imidoyl moiety to afford the corresponding 2-chloroquinolines. Facile syntheses of nine 2-chloroquinoline derivatives from several anilines and their postulate mechanism is described. The postulate mechanism of 2-chloroquinoline formation via imidoyl moiety as a good leaving group shows that the reaction consists of the following three steps: (1) generation of phenylisocyanate, (2) quinoline ring formation, and (3) chlorination on C2 position of quinoline.

Journal Article↗
PubMed2002

Study of the ligand substitution reactions of cis,cis-[(bpy)(2)(L)RuORu(L')(bpy)(2)](n+) (L, L' = H(2)O, OH(-), NH(3)) using electrospray ionization mass spectrometry and (1)H NMR.

We have successfully applied electrospray ionization mass spectrometry (ESI-MS) and (1)H NMR analyses to study ligand substitution reactions of mu-oxo ruthenium bipyridine dimers cis,cis-[(bpy)(2)(L)RuORu(L')(bpy)(2)](n+) (bpy = 2,2'-bipyridine; L and L' = NH(3), H(2)O, and HO(-)) with solvent molecules, that is, acetonitrile, methanol, and acetone. The results clearly show that the ammine ligand is very stable and was not substituted by any solvents, while the aqua ligand was rapidly substituted by all the solvents. In acetonitrile and acetone solutions, the substitution reaction of the aqua ligand(s) competed with a deprotonation reaction from the ligand. The hydroxyl ligand was not substituted by acetonitrile or acetone, but it exchanged slowly with CH(3)O(-) in methanol. The substitution reaction of the aqua ligands in [(bpy)(2)(H(2)O)Ru(III)ORu(III)(H(2)O)(bpy)(2)](4+) was more rapid than that of the hydroxyl ligand in [(bpy)(2)(H(2)O)Ru(III)ORu(IV)(OH)(bpy)(2)](4+). In methanol, slow reduction of Ru(III) to Ru(II) was observed in all the mu-oxo dimers, and the Ru-O-Ru bridge was then cleaved to give mononuclear Ru(II) complexes.

Journal Article↗
PubMed2001

Structure of a variant of lac repressor with increased thermostability and decreased affinity for operator.

A single amino acid substitution, K84L, in the Escherichia coli lac repressor produces a protein that has substantially increased stability compared to wild-type. However, despite the increased stability, this altered tetrameric repressor has a tenfold reduced affinity for operator and greatly decreased rate-constants of inducer binding as well as a reduced phenotypic response to inducer in vivo. To understand the dramatic increase in stability and altered functional properties, we have determined the X-ray crystal structures of a dimeric repressor with and without the K84L substitution at resolutions of 1.7 and 3.0 A, respectively. In the wild-type dimer, K84-11, Lys84 forms electrostatic interactions at the monomer-monomer interface and is partially exposed to solvent. In the K84L-11 substituted protein there is reorientation of the N-subdomains, which allows the leucine to become deeply buried at the monomer-monomer interface. This reorientation of the N-subdomains, in turn, results in an alteration of hydrogen bonding, ion pairing, and van der Waals interactions at the monomer-monomer interface. The lysine residue at position 84 appears to exert its key effects by destabilizing the "optimal" conformation of the repressor, effectively loosening the dimer interface and allowing the repressor to adopt the conformations necessary to function as a molecular switch.

Amino Acid Substitution↗
PubMed2005

Obtaining the structure and bond rotational potential of a substituted ethane by NMR spectroscopy of solutions in nematic liquid-crystalline solvents.

Partially averaged dipolar couplings (also referred to as residual dipolar couplings) D(ij) can be obtained from the analysis of the NMR spectra of molecules dissolved in liquid-crystalline solvents. Their values for a nonrigid molecule depend upon the bond lengths and angles, the rotational potentials, and the orientational order of the molecules. The molecule studied, 1-chloro-2-bromoethane, is one of the simplest example of a substituted alkane in which the rotational potential has three minimum-energy positions, trans and gauche+/-conformations, and the present investigation explores the problems inherent in deriving the form of the potential and the molecular geometry from the set of partially averaged couplings between the protons, and between protons and (13)C nuclei. The geometrical parameters and the rotational potential obtained are compared with the results from a density-functional theory method.

Journal Article↗
PubMed1997

Formation of Bromocarbenium Bromide Ion Pairs in the Electrophilic Bromination of Highly Reactive Olefins in Chlorinated Aprotic Solvents.

The kinetics and the products of bromination of several substituted stilbenes with tetrabutylammonium tribromide (TBAT) have been investigated in aprotic solvents at different temperatures. Stilbenes bearing electron-withdrawing or moderately electron-donating substituents gave stereospecifically the anti addition products. The reactions followed a second-order rate law, and an inverse kinetic isotope effect (KIE), k(H)/k(D) = 0.85(0.05), was found for the bromination of cis-stilbene. The reactions of cis- and trans-4,4-dimethoxystilbenes yielded mixtures of meso and d,l dibromides both in chloroform and 1,2-dichloroethane. The rate constants (k(Br)()3()-) measured for the latter olefins deviated considerably from the Hammett correlations, and added bromide had a significant effect on the rates. The reactions of these activated stilbenes with molecular Br(2), carried out at low Br(2) concentration, followed a mixed second/third-order rate law. The kinetic and product distribution data for the reaction, with TBAT, of stilbenes bearing electron-withdrawing or moderately electron-donating substituents are interpreted on the basis of the known mechanism involving a product- and rate-determining nucleophilic attack by bromide on the olefin-Br(2) pi-complex. The data related to the bromination of the more activated methoxystilbenes are rationalized considering that, for these olefins, even in aprotic solvents, the ionization of the initially formed 1:1 pi-complex to a bromocarbenium bromide ion pair can compete both with the formation of a bromonium-tribromide ion pair and with the nucleophilic attack by Br(-). For this second-order process (first order in Br(2)), the kinetic constants and the activation parameters have been measured in chloroform and 1,2-dichloroethane and the activation parameters have been compared with those related to the third-order Br(2) addition and to the reaction with TBAT.

Journal Article↗
PubMed2002

Electronic effects and the stereochemistries in rearrangement-displacement reactions of triaryl(halomethyl)silanes with fluoride and with alkoxide ions.

Tetrabutylammonium fluoride (TBAF) reacts with (halomethyl)diphenyl(para-substituted-phenyl)silanes (13, X = Cl), 14 (X = Br), and 15 (X = I) in ether solvents to give fluorodiphenyl(para-substituted-phenylmethyl)silanes (17a) and fluorophenyl(phenylmethyl)(para-substituted-phenyl)silanes (20a) by attack on silicon and migrations of the phenyl or the para-substituted-phenyl groups to C-1 with displacement of chloride ion. Sodium methoxide in dioxane effects rearrangement-displacements of 14 (X = Br) to yield methoxydiphenyl(para-substituted-phenylmethyl)silanes (17b) and methoxyphenyl(phenylmethyl)(para-substituted-phenyl)silanes (20b). The migratory aptitudes of the varied phenyl groups in rearrangement-displacements of 13 with F(-) at 25 degrees C are p-CF(3)-Ph, 2.72 > p-Cl-Ph, 1.67 > Ph, 1.00 > p-CH(3)-Ph, 0.91 > p-CH(3)O-Ph, 0.58 > p-(CH(3))(2)N-Ph, 0.55. For reactions of 14 with sodium methoxide in dioxane, the migratory aptitudes at 23 degrees C are p-CF(3)-Ph, 2.53 > p-Cl-Ph, 1.64 > Ph, 1.00 > p-CH(3)O-Ph, 0.84 > p-CH(5)-Ph, 0.79 > p-(CH(3))(2)N-Ph, 0.68. The migratory aptitudes in the above rearrangement-displacements are increased by electron-withdrawing substituents, and logarithms of the migratory aptitudes give satisfactory linear correlations with sigma and/or sigma-zero values of the phenyl substituents. Hammett correlations however of the migratory aptitudes from reactions of F(-) with 13 (X = Cl) at 0 and -20 degrees C, 14 (X = Br) at 23, 0, and -20 degrees C, and 15 (X = I) at 23 degrees C are not linear. (+)-(Bromomethyl)methyl-1-naphthylphenylsilane (23, +8.29 degrees, cyclohexane) reacts with CsF and with TBAF in THF to give benzylfluoromethyl-1-naphthylsilane (51, = 0.00 degrees, cyclohexane) and fluoromethyl-(1-naphthylmethyl)phenylsilane (52, impure) in 10.4:1 ratio along with unchanged 23 ( 8.29 degrees, cyclohexane). Sodium methoxide and (+)-23 in dioxane at 25 degrees C and at 0 degrees C yield (+)-benzylmethoxymethyl-1-naphthylsilane (64) and (+)-methoxymethyl(1-naphthylmethyl)phenylsilane (65) in approximately 9:1 ratio. The conversions of (+)-23 to (+)-64 occur with >93% inversion about silicon. Reaction of (+)-23 with sodium methoxide at 25 degrees C to give (+)-65 also occurs with inversion. Further, sodium ethoxide and sodium 2-propoxide react with (+)-23 at 20-25 degrees C by rearrangement-displacements on silicon with phenyl migrations to yield (+)-benzylethoxymethyl-1-naphthylsilane (69) and (+)-benzylmethyl-1-naphthyl-2-propoxysilane (70), respectively, each with >95% inversion about silicon. The mechanisms of rearrangement-displacements of 13-15 and (+)-23 by fluoride and by alkoxide ions are discussed.

Journal Article↗
PubMed1977

Solvent denaturation of globular proteins: unfolding by the monoalkyl- and dialkyl-substituted formamides and ureas.

The effects of the monoalkyl and dialkyl-substituted formamide series of denaturants on the native conformation of sperm whale myoglobin, horse heart cytochrome c, and Glycera dibranciata (single chain) hemoglobin have been investigated by spectral measurements in the Soret region (409 and 422 nm) and optical rotation measurements (265nm). The effectiveness of these two classes of protein denaturants is similar to the other straight-chain compounds of the urea, amide, and alcohol classes, examined in previous investigations from our laboratory. Their denaturing effectiveness is found to increase with increasing chain length or hydrocarbon content of the substituent alkyl groups. Application of the Peller and Flory equation to the denaturation data of the formamides shows that both the polar and the nonpolar group contributions to the protein-denaturant interactions have to be taken into account in order to correctly predict the observed denaturation midpoints. Additivity of the hydrophobic, KHø, and the polar, Kp, group contributions to the binding constants, KB = nKHø + Kp, with n = 1 or 2 for the mono- of the di-alkyl substituted denaturants gave best account of the experimental data. The KHø values used were based on free energy transfer data of various alkyl groups or the Scheraga-Nemethy theory of hydrophobic bonding. The assumption of group contributions of the denaturant to KB were also applied to the denaturation data of the unsubstituted amides and some examples of the monoalkyl and symmetrically substituted dialkyl ureas, taken from the literature.

Cytochrome c Group↗