Circular dichroism and the conformational properties of soybean beta-amylase

The conformational properties of soybean β-amylase were investigated by the circular dichroism probe and measurement of enzyme activity. The enzyme exhibited a positive circular dichroism band at 192 nm, a negative band at 222 nm, and a shoulder near 210 nm. Analysis of the spectrum in the far ultraviolet zone indicated the presence of approximately 30% of α helix and 5–10% of β-pleated sheet, the rest of the polypeptide main chain possessing aperiodic structure. In the near ultraviolet reagion, the enzyme protein showed at least six positive peaks at 259, 265, 273, 281, 292, and 297 nm. The positive bands at 292 and 297 nm remained unaltered on acetylation of the enzyme by N-acetylimidazole and were assigned to tryptophanyl chromophores. These bands were affected in intensity in the presence of maltose or cycloheptaamylose, which indicates that some tryptophan residues are situated at the binding sites. The native conformation of soybean β-amylase was found to be sensitive to pH variation (below pH 5 and above pH 10), sodium dodecyl sulfate, guanidine hydrochloride, and heating to 50–55 °C. Complete disorganization of the secondary structure was attained by 6 m guanidine hydrochloride. Sodium dodecyl sulfate was effective in disturbing the tertiary structure of the enzyme but did not affect significantly the secondary structure. Enzymatic inactivation was paralleled by the decrease of circular dichroism bands in the near ultraviolet region as produced by the denaturants. It is concluded that the uniquely folded structure of the enzyme contains some less rigid domains and a rigid core stabilized by hydrophobic interactions, electrostatic interactions, and hydrogen bonds.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unusual effect of salts on the homodimeric structure of NADH oxidase from Thermus thermophilus in acidic pH

The unusual salt-dependent behavior of the homodimeric flavoenzyme NADH oxidase from Thermus thermophilus in acidic pH has been studied using circular dichroism (CD) and sedimentation velocity. The native-like secondary and quaternary structures in acidic low ionic strength conditions were significantly perturbed by the addition of salts. The peptide region of the CD spectra showed a major salt-induced conformational change in the protein secondary structure. Sedimentation velocity experiments showed dissociation of the homodimeric structure of NADH oxidase in the presence of salt (>1 M). The new acidic conformation of the protein was stabilized by high ionic strength as indicated by a salt-induced increase in the melting temperature of the protein, and by a shift in the apparent pK(a) values of the conformational transition to a less acidic pH. Distortion of the dominant alpha-helical signal was expressed as the disappearance of the parallel polarized Moffitt exciton band at 208 nm without an accompanying loss of amplitude of n-->pi* electronic transitions at 222 nm. The unusual CD spectra correlated qualitatively with the theoretically calculated CD spectra of short alpha-helical structures and/or twisted beta-sheets. Differences between the experimentally obtained CD spectra and theoretical calculations (AGADIR) of the alpha-helical content of NADH oxidase indicate a role for non-local interactions in the protein conformation at high ionic strength and low pH. These findings indicate the importance of the homodimeric interface and electrostatic interactions for maintaining the structural integrity of this thermophilic protein.     

The amyloid precursor protein interacts with neutral lipids.

The amyloid protein precursor (APP) was incorporated into liposomes or phospholipid monolayers. APP insertion into liposomes required neutral lipids, such as L-alpha-phosphatidylcholine, in the target membrane. It was prevented in vesicles containing L-alpha-phosphatidylserine. The insertion was enhanced in acidic solutions, suggesting that it is modulated by specific charge/charge interactions. Surface-active properties and behaviour of APP were characterized during insertion of the protein in monomolecular films of L-alpha-phosphatidylcholine, L-alpha-phosphatidylethanolamine or L-alpha-phosphatidylserine. The presence of the lipid film enhanced the rate of adsorption of the protein at the interface, and the increase in surface pressure was consistent with APP penetrating the lipid film. The adsorption of APP on the lipid monolayers displayed a significant head group dependency, suggesting that the changes in surface pressure produced by the protein were probably affected by electrostatic interactions with the lipid layers. Our results indicate that the penetration of the protein into the lipid monolayer is also influenced by the hydrophobic interactions between APP and the lipid. CD spectra showed that a large proportion of the alpha-helical secondary structure of APP remained preserved over the pH or ionic strength ranges used. Our findings suggest that APP/membrane interactions are mediated by the lipid composition and depend on both electrostatic and hydrophobic effects, and that the variations observed are not due to major secondary structural changes in APP. These observations may be related to the partitioning of APP into membrane microdomains.     

pH dependence of the circular dichroic bands of phenylmethanesulfonyl-mesentericopeptidase.

The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl-mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2--10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH-dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the "exposed" tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.     

Conformational studies of NADPH cytochrome P-450 reductase by circular dichroism: interaction with phospholipids

Ultraviolet circular dichroism spectrum of purified NADPH cytochrome P-450 reductase was characterized by two negative bands centered at 208 and 222 nm. The approximation of the alpha-helical content from the value of the mean residue ellipticity at 222 nm indicated 28% of alpha-helical structures. Heat inactivation of the enzyme was associated to a drastic change in the secondary structure of the protein. Membrane reconstitution experiments by inclusion of the enzyme into liposomes revealed that the conformation of NADPH cytochrome P-450 reductase was sensitive to its phospholipid environment. Egg lecithin as well as synthetic phosphatidylcholines, at the optimal phospholipid-enzyme molar ratio 200, was able to increase up to 37% the mean residue ellipticity at 222 nm. Addition of phosphatidylserine or phosphatidylethanolamine produced no effect. Non-ionic detergent such as Emulgen 913 weakly enhanced the mean residue ellipticity.     

Further studies of detergent-induced conformational transitions in proteins. Circular dichroism of ovalbumin, bacterial alpha-amylase, papain, and beta-lactoglobulin at various pH values.

The conformational transitions of ovalbumin, bacterial α-amylase, papain, and β-lactoglobulin were studied in the absence and presence of sodium dodecyl sulfate (SDS) between pH 2.75 and 12.0 by means of circular dichroism (CD) measurement. The weight ratios of SDS to protein in solutions were 14:1 in all experiments. The CD bands in the near-ultraviolet spectral region were strongly reduced by SDS, whereas those in the far-ultraviolet were enhanced. With the exception of the amylase, the mean residue ellipticities of the proteins at 222 nm were increased by SDS, especially in acidic solutions. At a pH of about 3.0, the [θ]₂₂₂ values approached ?17 (±2) · 10³ deg · cm² · dmol^?1. It is assumed that at a sufficiently low pH value the proteins which are complexed with SDS have a similar backbone conformation of moderate helical content. In alkaline solutions, the detergent effect was largely reduced due to electrostatic repulsion between the negatively charged protein and dodecyl ions. The near-ultraviolet spectra of ovalbumin, papain, and β-lactoglobulin at pH 6.4 were analyzed. Assignment of the resolved bands to the appropriate chromophores was also attempted.     

Effect of acidic phospholipids on the structural properties of recombinant cytosolic human glyoxalase II

A peculiar characteristic of highly concentrated cytosolic recombinant human glyoxalase II (GII) solutions is to undergo partial precipitation. Previous work indicated that anionic phospholipids (PLs) exert a noncompetitive inhibition on the enzymatic activity of the soluble enzyme. In this study, FTIR spectroscopy was used to analyze the structural properties and the thermal stability of the soluble protein in the absence and in the presence of liposomes made of different phospholipids (PLs). The structural analysis was performed on the precipitate as well. The interaction of acidic PLs with GII lowered the thermal stability of the enzyme and inhibited protein intermolecular interactions (aggregation) brought about by thermal denaturation. Infrared data indicated that ionic and hydrophobic interactions occur between GII and acidic PLs causing small changes in the secondary structure of the enzyme. No interactions of the protein with egg phosphatidylcholine liposomes were detected. The results are consistent with the destabilization of the protein tertiary structure, and indicate that GII possesses hydrophobic part(s) that interact with the acyl chains of PLs. Data on precipitated GII did not show remarkable modification of secondary structure, suggesting that hydrophobic stretches of the enzyme may also be involved in the protein-protein association (precipitation) at high GII concentration. The alterations in the GII structure and the noncompetitive inhibition exerted by acidic PLs are strictly related.     

Incorporation of bovine thyroid peroxidase in liposomes

Bovine thyroid peroxidase (TPO), an enzyme requiring lipids for demonstrating catalytic activity, was incorporated in liposomes made of pure phospholipids. The enzyme did not show high differences in activity when bilayer thickness was changed, but dipalmitoyl phosphatidyl choline (DPPC) seemed to be more appropiate for activity. The perturbation caused on lipid fluidity by enzyme incorporation was studied by differential scanning calorimetry (DSC) and fluorescence polarization of the apolar probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The complexes of TPO with dimyristoyl phosphatidyl choline (DMPC), DPPC, and distearoyl phosphatidyl choline (DSPC) bilayers showed transition temperatures (T_c) which were lower than the characteristic ones shown by liposomes with the respective phospholipids alone. The microsomal fraction from which TPO was extracted was in the fluid state at 37°C, the temperature at which thyroid peroxidase works ‘in vivo’. Since the effect of the protein in lowering the transition temperature of the phospholipids was so low, the contribution of phospholipids containing unsaturated fatty acids has to be essential for obtaining a fluid bilayer at body temperature.     

Effects of membrane perturbants on UDP-glucuronosyltransferase activity in rat-liver microsomes. Circular dichroism studies.

Rat-liver microsomes were treated with two non-ionic detergents, Triton X-100 and Lubrol WX, with phospholipase A2, or with aqueous acetone solution. The activity of the membrane-bound UDP-glucoronosyltransferase (UDPGT, EC 2.4.1.17) was measured after the treatment with these perturbants. At the same time, modifications of the secondary structure of the microsomal proteins were followed and studied by circular dichroism (CD) spectroscopy. The detergents greatly activated UDPGT, maximally at a 1 mM concentration of either detergent. The maximally activating Triton X-100 treatment did not greatly change the ellipticity of the microsomes at 222 nm ((theta)222), whereas that with Lubrol WX affected the secondary structure of the membrane proteins more strongly. UDPGT activation also occurred in phospholipase A2-treated microsomes. Maximal activation was obtained after 1--5 min of incubation and was stable throughout the experiment. Phospholipase A2 at the ratio of microsomal protein to phospholipase 250 : 1 (w/w) slightly increased (theta)222 after 10 min of incubation and did not change it further even after 30 min of incubation. Treatment of liver microsomes with a 10 : 90 (v/v) aqueous acetone solution removed 90% of the total membrane phospholipids, particularly phosphatidylcholine and phosphatidylethanolamine. The UDPGT activity was decreased in lipid-depleted microsomes, and the enzyme was not reactivated when phosphatidylcholine-lysophosphatidylcholine liposomes were added at a low temperature. An even greater decrease was obtained when the lipid binding was carried out at 37 degree C. Lipid-depleted microsomes had a high (theta)222 associated with a red-shift of 2 nm, indicating partial aggregation of membrane proteins and an increase in the alpha-helical content of the protein after acetone extraction. However, this particular protein structure was partially reversible, since a binding of phospholipids to lipid-depleted microsomes gave a (theta)222 close to that found in control microsomes. The UDPGT activity was not dependent on the secondary structure of the membrane proteins.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

DOTAP/DOPE and DC-Chol/DOPE lipoplexes for gene delivery studied by circular dichroism and other biophysical techniques

Cationic liposomes give rise to stable complexes with DNA molecules (lipoplexes) that are of great interest for gene delivery applications. In particular, liposomes made up by a cationic lipid (DOTAP or DC-Chol) and a zwitterionic lipid (DOPE), produce stable adducts with single and double-stranded DNA oligonucleotides. Formation of these lipoplexes has been further addressed here by circular dichroism spectroscopy (CD) and by other independent biophysical methods. Titration of DNA oligonucleotides with cationic liposomes resulted into significant modifications of their circular dichroic bands. Such spectral modifications were ascribed to progressive DNA condensation and loss of native conformation, as a consequence of the electrostatic interactions taking place between the phosphate groups of DNA and the positively charged head groups of cationic lipids. In all cases, the loss of the CD feature characteristic of the native DNA conformation closely matched the inflection point of Zeta potential profiles. The resulting adducts showed peculiar and non-canonical CD spectra, while exhibiting appreciable stability at physiological pH.     

Phospholipids stabilize the secondary structure of the sodium-coupled branched-chain amino acid carrier of Pseudomonas aeruginosa

For functional reconstitution of bacterial cotransporters (carriers or permeases) including the sodium-coupled branched-chain amino acid carrier (LIV-II carrier) of Pseudomonas aeruginosa, the presence of phospholipid is required through the process of solubilization and purification of the transporters from the bacterial membranes, suggesting the possibility that phospholipid may stabilize the structure of the cotransporter proteins to be in a functional form. In this study, this possibility was examined by studying the effect of denaturant on the secondary structure of the LIV-II carrier purified in the absence and presence of phospholipid using circular dichroism (CD) spectroscopy. CD spectra of the purified LIV-II carrier solubilized in n-octyl-beta-D-glucopyranoside (OG), OG/dioleoylphosphatidylethanolamine (DOPE)/dioleoylphosphatidylglycerol (DOPG) mixture, and dispersed into DOPE/DOPG small unilamellar vesicles were measured in the absence of denaturant. The three spectra were very similar and had a trough at 222 nm with mean residue molar ellipticity of -23000 deg.cm(2)/dmol and a shoulder at 208 nm. CD spectral analyses with three different methods (S.W. Provencher, J. Gl?ckner, Estimation of globular protein secondary structure from circular dichroism, Biochemistry 20 (1981) 33-37; J.Y. Yang, C.-S.C. Wu, H.Z. Martinez, Calculation of protein conformation from circular dichroism, Methods Enzymol. 130 (1986) 208-269; N. Sreerama, R.W. Woody, A self-consistent method for the analysis of protein secondary structure from circular dichroism, Anal. Biochem. 209 (1993) 32-44) revealed that the LIV-II carrier solubilized in OG/DOPE/DOPG mixture contained 69-75% alpha-helix and 0-9% beta-sheet. Addition of 6 M guanidine hydrochloride decreased 48% of the amplitude at 222 nm of the CD spectrum of the carrier solubilized in OG alone and 9-14% of the CD amplitude of the carrier solubilized in OG/DOPE/DOPG or OG/dioleoylphosphatidylcholine mixture and dispersed in liposomes composed of DOPE/DOPG. These results show that the ordered secondary structure of the LIV-II carrier is partially unfolded in OG without phospholipid by denaturant but is greatly stabilized with phospholipids with oleoyl chains independently of their polar head group composition and suggest that the alpha-helical structure of the carrier is mainly embedded in the lipid environment.     

Electrostatic and hydrophobic forces tether the proximal region of the angiotensin II receptor (AT1A) carboxyl terminus to anionic lipids

The carboxyl terminus of the type-1 angiotensin II receptor (AT(1A)) is a focal point for receptor activation and deactivation. Synthetic peptides corresponding to the membrane-proximal, first 20 amino acids of the carboxyl terminus adopt an alpha-helical conformation in organic solvents, suggesting that the secondary structure of this region may be sensitive to hydrophobic environments. Using surface plasmon resonance, immobilized lipid chromatography, and circular dichroism, we examined whether this positively charged, amphipathic alpha-helical region of the AT(1A) receptor can interact with lipid components in the cell membrane and thereby modulate local receptor attachment and structure. A synthetic peptide corresponding to the proximal region of the AT(1A) receptor carboxyl terminus (Leu(305) to Lys(325)) was shown by surface plasmon resonance to bind with high affinity to the negatively charged lipid, dimyristoyl L-alpha-phosphatidyl-DL-glycerol (DMPG), but poorly to the zwitterionic lipid, dimyristoyl L-alpha-phosphatidylcholine (DMPC). In contrast, a peptide analogue possessing substitutions at four lysine residues (corresponding to Lys(307,308,310,311)) displayed poor association with either lipid, indicating a crucial anionic component to the interaction. Circular dichroism analysis revealed that both the wild-type and substituted peptides possessed alpha-helical propensity in methanol and trifluoroethanol, while the wild-type peptide also adopted partially inserted helical structure in DMPG and DMPC liposomes. In contrast, the substituted peptide exhibited spectra that suggested the presence of beta-sheet and alpha-helical structure in both liposomes. Immobilized lipid chromatography was used to characterize the hydrophobic component of the membrane interaction, and the results demonstrated that hydrophobic and electrostatic interactions mediated the binding of the wild-type peptide but that the substituted peptide bound to the model membranes mainly via hydrophobic forces. We propose that, in intact AT(1A) receptors, the proximal carboxyl terminus associates with the cytoplasmic face of the cell membrane via a high-affinity, anionic phospholipid-specific tethering that serves to increase the amphipathic helicity of this region. Such associations may be important for receptor function and common for G protein-coupled receptors.     

Acid denaturation of recombinant porcine growth hormone: formation and self-association of folding intermediates

We have investigated the conformational changes incurred during the acid-induced unfolding and self-association of recombinant porcine growth hormone (pGH). Acidification (pH 8 to pH 2) of pGH resulted in intrinsic fluorescence, UV absorbance, and near-UV CD transitions centered at pH 4.10. At pH 2.0, a red shift in the fluorescence emission maximum of approximately 3 nm and a 15% loss of the far-UV CD signal at 222 nm imply that the protein did not become extensively unfolded. Acidification in the presence of 4 M urea resulted in similar pH-dependent transitions. However, these occurred at a higher pH (approximately 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the fluorescence emission maximum suggests that unfolding was greater than in the absence of urea. The presence of a prominent peak centered at 298 nm in the near-UV CD spectrum, which is absent without urea, signifies further differences in the intermediates generated at pH 2. Sedimentation equilibrium experiments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-association was strongly promoted at pH 2 while urea reduced self-association at both pH 8 and pH 2. These results demonstrate that acidification of pGH in the absence or presence of 4 M urea induced the formation of molten globule-like states with measurable differences in conformation. Similarities and differences in these structural conformations with respect to other growth hormones are discussed.     

Interaction of rat brain cytidylate cyclase with phospholipids

The interaction of rat brain cytidylate cyclase with some phospholipids such as L-alpha-phosphatidylcholine (PC), L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylethanolamine (PE) and L-alpha-phosphatidic acid (PA) was studied. Cytidylate cyclase activity of Triton X-100 - solubilized fraction was inhibited by PS, PE and PA, but not with PC. The addition of PC to the incubation mixture containing PS, PE or PA dose - dependently reversed the inhibition of enzyme activity by these phospholipids. Phospholipids showed similar effect on the intact membrane - bound enzyme. PC could reactivate the enzyme which was inactivated by deoxycholate treatment, suggesting that PC may be an important factor to reconstitute an active conformation of the enzyme. These findings indicate that cytidylate cyclase could be regulated by phospholipids constituting its microenvironment of the membrane.     

Cloning, expression, purification, and characterization of a novel epoxide hydrolase from Aspergillus niger SQ-6

A novel epoxide hydrolase from Aspergillus niger SQ-6 has now been cloned by inverse PCR. Its gene shows eight exons including a non-coding exon at its 5'-terminal (GenBank Accession No. AY966486). Phylogenetic analysis using deduced amino acid sequence (395 aa) confirms it as an epoxide hydrolase and shares 58.3% identity with that of A. niger LCP521 (GenBank Accession No. AF238460). The predicted catalytic triad is composed of Asp(191), His(369) and Glu(343). Active recombinant epoxide hydrolase has been successfully expressed in Escherichia coli as protein fusions with a poly-His tail. Scale-up fermentation can yield 2.5g/L of recombinant protein. The electrophoretic pure recombinant protein, which shows similar characterization as natural enzyme purified from A. niger SQ-6, can be easily purified by Ni(2+)-chelated affinity and gel-filtration chromatography. Optimal pH and temperature for purified enzyme are pH 7.5 and 37 degrees C, respectively. The K(m), k(cat) and maximal velocity (V(max)) for p-nitrostyrene oxide are determined to be 1.02mM, 172s(-1) and 231micromol min(-1)mg(-1), respectively. The enzyme can be inhibited by oxidant (H(2)O(2)), solvent (Tetrahydrofuran) and several metal ions including Hg(2+), Fe(2+) and Co(2+). This (R)-stereospecific epoxide hydrolase exhibits high enantioselectivity (enantiomeric excess value, 99%) for the less hindered carbon atom of epoxide. It may be an industrial biocatalyst for the preparation of enantiopure epoxides or vicinal diols.     

Overexpression of Arabidopsis thaliana soluble epoxide hydrolase 1 in Pichia pastoris and characterisation of the recombinant enzyme

Epoxide hydrolases are enzymes involved in metabolism and defense of plants. Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana. To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed. We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis. We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A. thaliana. A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose. The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis. The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively. The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C. Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively. The activity was 50-fold lower towards cis-stilbene oxide. The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.     

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.     

Analysis of the conformation and stability of Escherichia coli derived recombinant human interleukin 4 by circular dichroism

The conformation and stability of Escherichia coli derived recombinant human interleukin 4 (rhuIL-4) have been examined by circular dichroism (CD). Protein unfolding was detected by ellipticity changes at 222 nm with increasing concentrations of guanidine hydrochloride (GdnHCl). The unfolding midpoint ([GdnHCl]1/2) was 3.7 M, the free energy of unfolding, (delta GDH2O), was 5.9 kcal/mol and the dependence of delta GD on the GdnHCl concentration (m) was 1.6 (kcal/mol)/M. This unfolding was demonstrated to be reversible upon removal of the GdnHCl by dialysis. Analysis of the far-UV CD spectrum indicated the presence of a high percentage of alpha-helical structure (ca. 73%). A small change in ellipticity was noted over the pH range 1.9-9.6, suggesting that the protein undergoes a minor conformational change with an apparent pKa of 4.17. Virtually complete biological activity, measured in vitro in a T-cell proliferation assay, was recovered following exposure to extreme values of pH (i.e., pH 3 and 10). An analysis of the near-UV CD spectrum indicated that the single tryptophan residue at position 91 was unconstrained and most likely exposed to the solvent. Titration with 4,4'-dithiodipyridine and 2-nitrothiosulfobenzoate established that the six cysteine residues in rhuIL-4 were involved in intramolecular disulfide linkages. These data support that rhuIL-4 has a highly stable three-dimensional structure.