Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Structure of amphotericin B aggregates as revealed by UV and CD spectroscopies

The aqueous and hydroalcoholic solutions of the heptenic macrolide amphotericin B display strong and variable signals in CD and absorption spectroscopies in the range of the π* ← π transition. An interpretation of the spectroscopic changes is proposed based on the equilibrium between two forms of the intermolecular organization: the aggregated one (A) with strong excitonic interaction and the nonaggregative one (B) whose spectra are like those of linear conjugated polyenes in true solution with a well-developed vibrational structure. The intermediate spectra are fitted by linear combination of the A- and B-form spectra. A two-level organization of the aggregates is proposed for the A-form: (1) a close packing of few molecules, which is the origin of the absorption maxima hypsochromic shift; and (2) interaction between the preceding small units inside the aggregates, which is spectroscopically expressed by the intense CD couplet.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Measuring enzyme hydration in nonpolar organic solvents using NMR

A very sensitive NMR method has been developed for measuring deuterated water bound to proteins suspended in nonpolar solvents. This has been used to determine the amount of bound water as a function of water activity for subtilisin Carlsberg suspended in hexane, benzene, and toluene and for alpha-chymotrypsin in hexane. The adsorption isotherms for subtilisin in the three solvents are very similar showing that water activity can be usefully employed to predict the amount of water bound to proteins in nonpolar organic media. Comparison of the degree of enzyme hydration reached in nonpolar solvents with that obtained in air shows that adsorption of strongly bound water is hardly affected by the low dielectric medium, but adsorption of loosely bound water is significantly reduced. This suggests that the hydrophobic regions of the protein surface are preferentially solvated by solvent molecules, and that in a nonpolar environment formation of a complete monolayer of water over the protein surface is thermodynamically unfavorable. (c) 1995 John Wiley & Sons, Inc.     

Correlation between catalytic activity and secondary structure of subtilisin dissolved in organic solvents

Fourier-transform infrared (FTIR) spectroscopy has been used to quantify the alpha-helix and beta-sheet contents of subtilisin Carlsberg dissolved in several nonaqueous, as well as aqueous, solvents. Independently, the catalytic activity of the enzyme has been measured in the same solvents. While our previous FTIR studies revealed no connection between the secondary structure and enzymatic activity for subtilisin suspended in various organic solvents, a very different situation is observed herein for the dissolved enzyme. Specifically, if either the alpha-helix or beta-sheet content in a given solvent is higher or lower than in water, no appreciable enzymatic catalysis is observed. Conversely, when the secondary structure of subtilisin dissolved in a given nonaqueous solvent is similar to that in water, so is the enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 485-491, 1997.     

New considerations of the Barclay-Butler rule and the behavior of water dissolved in organic solvents

The Barclay–Butler (B-B) rule, which states that a linear relationship exists between the standard ΔH_vap and ΔS_vap for simple, non-associated liquids and their solutions, has been used to distinguish associated (‘abnormal’) liquids from simple (‘normal’) liquids. The exact character of the B-B plots depends on the standard states chosen for the liquid/solution and vapor. We examine the effects of using number density for both vapor and liquid states for pure liquids, non-aqueous solutions, aqueous solutions and solutions in which water is the solute. The utility of B-B plots to detect solute-induced order is strengthened, and we also find remarkable changes in the modified B-B relationship: (1) the points for small, H-bonded liquids, including water, are pulled below the general B-B line; (2) many solutions containing small, simple solutes have negative entropies of vaporization; and (3) solutions of water in several organic solvents, relevant to studies of proteins and micelles, appear ‘abnormal’.     

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.     

A simple and effective NMR cell for studies of encapsulated proteins dissolved in low viscosity solvents

Application of triple-resonance and isotope-edited-NOE methods to the study of increasingly larger macromolecules and their complexes remains a central goal of solution NMR spectroscopy. The slow reorientational motion of larger molecules leads to rapid transverse relaxation and results in losses in both resolution and sensitivity of multidimensional-multinuclear solution NMR experiments. A recently described technique employs a physical approach to increase the tumbling rate of macromolecules in an attempt to preserve access to the full range of structural restraints available to studies of smaller systems. This technique involves encapsulation of a hydrated protein in a surfactant shell which is subsequently solubilized in a low viscosity solvent. A simple, efficient and cost effective NMR cell that accommodates the moderate liquefaction pressures required in the encapsulation method is described. Application of the method to the 56 kD triose phosphate isomerase homodimer is demonstrated.     

Thermodynamics of the lipase-catalyzed esterification of glycerol and n-octanoic acid in organic solvents and in the neat reaction mixture

The thermodynamics of the lipase-catalyzed esterification of glycerol with n-octanoic acid have been investigated with acetonitrile, benzene, and toluene as solvents and in the neat reaction mixture (no organic solvent added). This esterification reaction leads to five products: 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol. This, in turn leads to a total of 12 reactions. Values of the equilibrium constants for these reactions have been measured (HPLC, GC, and LC/MS) at 37°C in the above mentioned media. The equilibrium constants range from 0.9 to 20.7, 0.20 to 8.0, 0.23 to 10.0, and 0.57 to 2.2 in acetonitrile, benzene, toluene, and neat media, respectively. Relative standard molar Gibbs free energies of formation Δ_fG_m⁰ of 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol in the organic solvents and in the neat reaction mixture have been calculated and used to compactly summarize the thermodynamics of these reactions. The results show an approximate correlation with the permittivities of the solvents.     

Active conformation of a-chymotrypsin in organic solvents as studied by circular dichroism

Circular dichroic (CD) spectra of a-chymotrypsin were measured in mixtures of water and ethanol, acetonitrile, or tetrahydrofuran. The catalytic activity of a-chymotrypsin in the different solvents was related to the change of CD bands especially that at 230 nm. The fine structure in near-UV region may also be useful to estimate the catalytic activity of a-chymotrypsin.     

Water-induced precipitation of cholesterol dissolved in organic solvents in the absence and presence of surfactants and salts

The precipitation of cholesterol dissolved in organic solvents, viz. methanol, ethanol, n-propanol, isopropanol, acetone and 1,4-dioxane, by the addition of water has been studied. The effects of the solvents towards the precipitation follow the order: methanol greater than ethanol greater than acetone greater than dioxane greater than n-propanol greater than iso-propanol, the solvent dioxane however exhibits a change in the order at higher concentration. Additives like Triton X-100, sodium cholate, sodium deoxycholate, sodium dehydro cholate, sodium salicylate and sodium chloride have some protective action against precipitation, the maximum protection being that of Triton X-100. The additives have shown better protective action in propanols and dioxane than in methanol, ethanol and acetone. Analysis of solvent composition and dielectric constant has revealed specific solvent effects on the water-induced precipitation of cholesterol. Thermodynamic analysis of the precipitation phenomenon and the unique role of solvent structure on cholesterol precipitation has been discussed.     

Thiolsubtilisin acts as an acetyltransferase in organic solvents

The catalytic mechanism of arylamine N-acetyltransferase has been proposed to involve Cys-His-Asp as its catalytic triad. Thiolsubtilisin, a chemically modified enzyme that has a catalytic triad of Cys-His-Asp at the active site, mimics the catalysis of arylamine N-acetyltransferase, serotonin N-acetyltransferase, histone N-acetyltransferase and amino acid N-acetyltransferase. Thiolsubtilisin not only can catalyze amino acid transacetylation, but is also able to catalyze amine transacetylation. Ethyl acetate was used as the acylating reagent to form N-acetyl amino acids and amines in organic solvents with moderate yield. Hence, these findings broaden our understanding of the structural features required for N-acetyltransferases activity as well as provide a structural relationship between cysteine protease and other N-acyltransferases.     

Study on the interaction of aromatic dyes with nucleic acids by means of UV, CD and NMR spectroscopies.

The interaction of several aromatic cationic dyes such as, ethidium bromide (EB), methylene blue (MB), acridine orange (AO), and Hoechst 33258 with calf-thymus DNA and poly(A)-poly(U) duplex was investigated. The different induced extrinsic Cotton effects (greater than 300 nm) were observed for DNA- and RNA-dye complexes. The binding properties of these complexes were examined by UV, CD, and NMR spectroscopies.     

Denaturation of bacteriorhodopsin by organic solvents

The denaturation of bacteriorhodopsin by various organic solvents was studied using absorption, circular dichroism (CD) and fluorescence measurements. Organic solvents with a hydrogen-bonding group caused the release of retinal. The CD measurements showed that the helical structure was maintained even in the denatured state, whereas its tertiary structure was destroyed. The change in fluorescence intensity of tryptophan and fluorescent retinal also confirmed that the tertiary structure was destroyed. Comparison of the denaturation efficiency of various organic solvents showed that the concentration at denaturation was inversely proportional to the partition coefficient of the denaturant. This inverse proportionality clearly indicated that denaturation was determined by the concentration of denaturants which partitioned into the hydrophobic region of the membrane. It was discussed from the experimental results that the tertiary structure of bacteriorhodopsin was stabilized by the hydrogen-bonding networks between side chains of the helices. The results obtained from analysis of the amino acid sequence were also consistent with the hydrogen-bonding mechanism for the formation of the tertiary structure.     

Interaction of a goose-type lysozyme with chitin oligosaccharides as determined by NMR spectroscopy

The interaction between a goose-type lysozyme from ostrich egg white (OEL) and chitin oligosaccharides [(GlcNAc)(n) (n = 2, 4 and 6)] was studied by nuclear magnetic resonance (NMR) spectroscopy. A stable isotope-labelled OEL was produced in Pichia pastoris, and backbone resonance assignments for the wild-type and an inactive mutant (E73A OEL) were achieved using modern multi-dimensional NMR techniques. NMR titration was performed with (GlcNAc)(n) for mapping the interaction sites of the individual oligosaccharides based on the shifts in the two-dimensional heteronuclear single quantum correlation (HSQC) resonances. In wild-type OEL, the interaction sites for (GlcNAc)(n) were basically similar to those determined by X-ray crystallography. In E73A OEL, however, the interaction sites were spread more widely over the substrate-binding cleft than expected, due to the multiple modes of binding. The association constant for E73A OEL and (GlcNAc)(6) calculated from the shifts in the Asp97 resonance (7.2 × 10(3) M(-1)) was comparable with that obtained by isothermal titration calorimetry (5.3 × 10(3) M(-1)). The interaction was enthalpy-driven as judged from the thermodynamic parameters (ΔH = -6.1 kcal/mol and TΔS = -1.0 kcal/mol). This study provided novel insights into the oligosaccharide binding mechanism and the catalytic residues of the enzymes belonging to family GH-23.     

Glucose condensation by glucoamylase in organic solvents

Summary Oligosaccharides were synthesized through the enzymatic condensation of D-glucose by glucoamylase in water-organic mixtures with high concentrations of two of diethylene glycol diethyl ether or triethylene glycol dimethyl ether. The effect of water content on the yield of reaction was studied; maximum yield was obtained with 10% (v/v) of water in the two systems. Kinetics of synthesis and products composition were different with the two solvents. 37% of glucose were condensed by action of glucoamylase from a reaction medium containing 20 g/L of glucose and 90% (v/v) of diethylene glycol diethyl ether.     

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.     

Characterization of the ground state dynamics of proteorhodopsin by NMR and optical spectroscopies

We characterized the dynamics of proteorhodopsin (PR), solubilized in diC7PC, a detergent micelle, by liquid-state NMR spectroscopy at T?=?323?K. Insights into the dynamics of PR at different time scales could be obtained and dynamic hot spots could be identified at distinct, functionally relevant regions of the protein, including the BC loop, the EF loop, the N-terminal part of helix F and the C-terminal part of helix G. We further characterize the dependence of the photocycle on different detergents (n-Dodecyl ??-D-maltoside DDM; 1,2-diheptanoyl-sn-glycero-3-phosphocholine diC7PC) by ultrafast time-resolved UV/VIS spectroscopy. While the photocycle intermediates of PR in diC7PC and DDM exhibit highly similar spectral characteristics, significant changes in the population of these intermediates are observed. In-situ NMR experiments have been applied to characterize structural changes during the photocycle. Light-induced chemical shift changes detected during the photocycle in diC7PC are very small, in line with the changes in the population of intermediates in the photocycle of proteorhodopsin in diC7PC, where the late O-intermediate populated in DDM is missing and the population is shifted towards an equilibrium of intermediates states (M, N, O) without accumulation of a single populated intermediate.