The thermophilic esterase from Archaeoglobus fulgidus: structure and conformational dynamics at high temperature

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

Conformational changes and stabilization induced by phosphate binding to 5′-methylthioadenosine phosphorylase from the thermophilic archaeon Sulfolobus solfataricus

The effect of phosphate, its analogues, and other substrates on structural features of recombinant 5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was investigated. Phosphate was found to exert a significant stabilizing effect on the protein against the inactivation caused by temperature, sodium dodecyl sulfate (SDS), urea, and proteolytic enzymes. In the presence of 100 mM phosphate: (i) the apparent transition temperature (Tm) of recombinant SsMTAP increased from 111 degrees to 118 degrees C; and (ii) the enzyme still retained 40% and 30% activity, respectively, after 30 min of incubation at 90 degrees C with 2% SDS or 8 M urea. The structure modification of SsMTAP by phosphate binding was probed by limited proteolysis with subtilisin and proteinase K and analysis of polypeptide fragments by SDS-PAGE. The binding of the phosphate substrate protected SsMTAP against protease inactivation, as proven by the disappearance of a previously accessible proteolytic cleavage site that was localized in the N-terminal region of the enzyme. The conformational changes of SsMTAP induced by phosphate and ribose-1-phosphate were analyzed by fluorescence spectroscopy, and modifications of the protein intrinsic fluorophore exposure, as a consequence of substrate binding, were evidenced.     

Solution structure of human saposin C in a detergent environment

Saposin C is a lysosomal, membrane-binding protein that acts as an activator for the hydrolysis of glucosylceramide by the enzyme glucocerebrosidase. We used high-resolution NMR to determine the three-dimensional solution structure of saposin C in the presence of the detergent sodium dodecyl sulfate (SDS). This structure provides the first representation of membrane bound saposin C at the atomic level. In the presence of SDS, the protein adopts an open conformation with an exposed hydrophobic pocket. In contrast, the previously reported NMR structure of saposin C in the absence of SDS is compact and contains a hydrophobic core that is not exposed to the solvent. NMR data indicate that the SDS molecules interact with the hydrophobic pocket. The structure of saposin C in the presence of SDS is very similar to a monomer in the saposin B homodimer structure. Their comparison reveals possible similarity in the type of protein/lipid interaction as well as structural components differentiating their quaternary structures and functional specificity.     

Sodium dodecyl sulfate activation of a plant polyphenoloxidase. Effect of sodium dodecyl sulfate on enzymatic and physical characteristics of purified broad bean polyphenoloxidase

Latent broad bean polyphenoloxidase was purified and shown to be activated by sodium dodecyl sulfate (SDS). Further characterization of the enzyme was carried out in the presence and absence of SDS. Activation of the enzyme increased in a sigmoidal manner with increasing SDS concentrations up to a maximum of 1.75 mM. The presence of SDS eliminated a low pH optimum induced by acid shocking. Increased thermolability of the enzyme was observed in the presence of SDS as well as an increased binding of [14C]dihydroxy-phenylalanine. Size exclusion chromatography on high performance liquid chromatography showed that the size and apparent molecular mass of the enzyme were slightly altered in the presence (48 kDa) versus absence (47 kDa) of SDS. Although the estimations were larger than those obtained by size exclusion techniques, no large differences in molecular weight were observed after sedimentation equilibrium of the enzyme in the presence (53.9 kDa) and absence (52.3 kDa) of SDS. Relative electrophoretic mobility and intrinsic fluorescence of tyrosine and tryptophan residues increased in a complex fashion as the SDS concentration was increased. Plateau regions in these latter experiments corresponded to concentrations of SDS needed for activation. The ability of SDS to activate the enzyme alters both its enzymatic and physical characteristics and suggests that a limited conformational change, due to binding of small amounts of SDS, may induce or initiate the activation of latent enzyme.     

Temperature-induced transitions in the conformation of intermediates in the hydrolytic cycle of myosin.

The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme-substrate complex and two different forms of enzyme-product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady-state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol-1 as well as thiol-2 groups. The long-lived ATPase intermediate occurring at temperatures above 10 degrees C adopts the same conformation with Mg2+ and Ca2+ ions. This intermediate does not protect the thiol-1 and thiol-2 groups but exposes a number of thiol-3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10 degrees C and is identical with that found in the presence of ADP at 0 degree C indicating a change in the rate-limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature-dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long-lived intermediate occurring above 10 degrees C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 degrees C. In the case of the monovalent-cation-stimulated ATPase a constant activation energy of around 70 kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0--35 degrees C.     

Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi

Spectroscopic techniques have been applied to investigate the conformation, local structure, and dynamic properties of the apoprotein of the lumazine protein from Photobacterium leiognathi and the holoprotein reconstituted with either the natural ligand 6,7-dimethyl-8-ribityllumazine or the closely related analogues riboflavin and 6-methyl-7-oxo-8-ribityllumazine (7-oxolumazine). The analogues are bound similarly to the natural prosthetic group. They exhibit similar shifts on binding in their absorption and fluorescence spectra, single-exponential fluorescence decays, and no independent motion from the protein as evident from a long-lived anisotropy decay (single-exponential phi = 10 ns, 20 degrees C) and high initial anisotropy. Steady-state anisotropy measurements result in similar KD's (40 nM, 20 degrees C, 50 mM inorganic phosphate) for all ligands. Circular dichroism in the far-UV region (190-250 nm) indicates no change in secondary structure on binding to the apoprotein. In the spectral region of 250-310 nm relatively large changes occur, indicating changes in the environment of the tyrosine and tryptophan residues. The single tryptophan residue shows a three-exponential decay of its fluorescence in both the apoprotein and the holoprotein. Radiationless energy transfer also occurs from the tryptophan to the bound ligand, especially evident with 7-oxolumazine. We have designed a new method for evaluation of the rate constant of energy transfer by measuring the (picosecond) rise time of the acceptor fluorescence. The anisotropy decay of the tryptophan residue shows two correlation times, a short one (phi approximately equal to 0.4 ns) representing rapid but restriced oscillation of this residue and a longer one (phi 2 = 5-7 ns, 20 degrees C) representing the motion of a larger segment of the protein.     

HIV-1 integrase catalytic core: molecular dynamics and simulated fluorescence decays.

Two molecular dynamics simulations have been carried out on the HIV-1 integrase catalytic core starting from fully determined crystal structures. During the first one, performed in the absence of divalent cation (6-ns long), the catalytic core took on two main conformations. The conformational transition occurs at approximately 3.4 ns. In contrast, during the second one, in the presence of Mg(2+) (4-ns long), there were no such changes. The molecular dynamics simulations were used to compute the fluorescence intensity decays emitted by the four tryptophan residues considered as the only chromophores. The decay was computed by following, frame by frame, the amount of chromophores that remained excited at a certain time after light absorption. The simulation took into account the quenching through electron transfer to the peptide bond and the fluorescence resonance energy transfer between the chromophores. The fit to the experimental intensity decays obtained at 5 degrees C and at 30 degrees C is very good. The fluorescence anisotropy decays were also simulated. Interestingly, the fit to the experimental anisotropy decay was excellent at 5 degrees C and rather poor at 30 degrees C. Various hypotheses such as dimerization and abnormal increase of uncorrelated internal motions are discussed.     

Escherichia coli OmpA retains a folded structure in the presence of sodium dodecyl sulfate due to a high kinetic barrier to unfolding.

Escherichia coli OmpA can be solubilized by sodium dodecyl sulfate (SDS) in its folded structure, and it unfolds upon heating. Although the heat-denatured OmpA remains unfolded after lowering the temperature, the addition of a non-ionic surfactant, octyl glucoside results in refolding of unfolded OmpA. In the present study, we investigated the refolding kinetics of OmpA in a mixed surfactant system of SDS and octyl glucoside using far- and near-UV circular dichroism and fluorescence spectroscopies. We found four kinetic phases in the refolding reaction, which logarithmically depended on the weight fraction of octyl glucoside. We also examined the unfolding kinetics of OmpA upon heating in the presence of SDS by temperature jump experiments. A comparison of the rate constants for the refolding and the unfolding reactions in SDS-only solution at 30 degrees C revealed that the folded form of OmpA in SDS solution is less stable than the unfolding form, and that the unfolding is virtually unobservable near room temperature due to a high kinetic barrier.     

Influence of heat and sodium dodecyl sulfate on the endopeptidase I from Bacillus sphaericus 9602

Endopeptidase I from Bacillus sphaericus is a stable enzyme which retains its activity at 37 degrees C in the presence of sodium dodecyl sulfate. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two forms of the enzyme: an active, fast-running form, for the enzyme preheated at 37 degrees C and a denatured, slow-running form, for the enzyme preheated at 100 degrees C. Such behavior is similar to that of the "heat-modifiable" outer membrane proteins from gram-negative bacteria. In the absence of sodium dodecyl sulfate, endopeptidase I aggregated in an enzymatically active dimer, with an apparent molecular weight of 90,000 daltons, which could be the native form of the enzyme.     

The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations

The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.     

Purification and Characterization of Aromatic L-Amino Acid Decarboxylase from Rat Kidney and Monoclonal Antibody to the Enzyme

Aromatic L-amino acid decarboxylase was purified from rat kidney to homogeneity, as judged by polyacrylamide gel electrophoresis, in the presence and absence of sodium dodecyl sulfate (SDS). The final preparation showed an activity of 3,4-dihydroxyphenylalanine (dopa) decarboxylation of approximately 11,000 nmol/min/mg of protein at 37 degrees C. The purified enzyme also catalyzed the decarboxylation of 5-hydroxytryptophan, tyrosine, tryptophan, and phenylalanine. The enzyme appeared to be composed of two identical subunits, each possessing a molecular weight of 48,000. The isoelectric point of the enzyme was estimated to be 6.7 in the presence of 8 M urea and 5.60-5.85 in its absence. To examine the identity of aromatic L-amino acid decarboxylase from various tissues, a monoclonal antibody directed against the enzyme from rat kidney was prepared. Immunotitration and analysis by antibody-affinity chromatography followed by SDS-polyacrylamide gel electrophoresis revealed that the enzymes from the striatum, adrenal medulla, pineal gland, liver, and kidney were indistinguishable with respect to immunological cross-reactivity and molecular size.     

Purification and characterization of 3 alpha-hydroxysteroid dehydrogenase from chicken hepatic cytosol

From the cytosol fraction (supernatant fluid at 105,000 g) of chicken liver, 3 alpha-hydroxysteroid dehydrogenase was purified to an apparently homogeneous state by differential precipitation with ammonium sulfate, followed by column chromatographies with DE 51, DEAE-Toyopearl, and Sephadex G-100. Finally the dehydrogenase was purified 103-fold on the basis of the cytosol fraction. Polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate (SDS) revealed that molecular weight of the purified enzyme was 66 kDa, while that of the native dehydrogenase in the absence of SDS was estimated as 660 kDa or more from the peak of the enzyme in elution profile from Sephacryl S-200 column chromatography. The dehydrogenase required NADPH specifically for reduction of 3-oxo group of 5 beta-androstanedione (Km = 1.6 microM). Optimal temperature for 3-oxo reduction was 50 C in incubation for 10 min.     

Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1

We examined the frequency-domain intensity decays of the intrinsic tryptophan fluorescence (Trp-59) from ribonuclease T1 (EC 3.1.27.3) (RNAase T1). At pH 5.5 in the native state (below 30 degrees C), the intensity decay of the single tryptophan residue is a single-exponential process. Conditions which result in protein unfolding were found to induce more complex intensity decays. At temperatures above 40 degrees C, or in the presence of guanidine hydrochloride, the intensity decays became obviously double exponential. In general, the main effect of temperature or guanidine was to induce a second subnanosecond component in the intensity decay. The increased complexity of the decays could not be explained by a unimodal distribution of decay times. These results indicate that conformational dispersion of protein structure can be one origin of the multi-exponential decays which are generally observed for protein fluorescence.     

Purification and characteristics of alkaline proteinase from alkalophylic Bacillus sp.]

Alkaline protease was purified from Bacillus sp. isolated from soil. The pH optimum was 11.5 at 37 degrees C. Calcium divalent cation was effective to stabilize the enzyme especially at higher temperatures. The proteolytic activity was inhibited by active site inhibitors of PMSF (Phenylmethylsulfonyl fluoride), and ions of Mg, Mn, Pb, Li, Zn, Ag, Hg. The enzyme was stable in the presence of some detergents, such as Triton-X-100, Tween-80, SDS (sodium dodecyl sulfate) and EDTA (ethylendiaminetetraacetic acid), pH 11.5 and 37 degrees C for 30 min. The optimum pH was 11.5 at 37 degrees C and the optimum temperature was 62 degrees C at pH 11.5.     

A recombinant glutamine-binding protein from Escherichia coli: effect of ligand-binding on protein conformational dynamics

We have investigated the effect of the binding of glutamine on the conformational dynamics of the recombinant glutamine binding protein (GlnBP) from Escherichia coli by steady-state and time-resolved fluorescence techniques. The structural stability of the protein was also studied by far-UV circular dichroism spectroscopy in the range of temperature between 25 and 80 degrees C. The results showed that the interaction of the protein with the ligand resulted in a marked change of the structural and conformational dynamics features of the protein. In particular, the fluorescence and circular dichroism data showed that the presence of glutamine resulted in a dramatic increase of the protein thermal stability of about 10 degrees C. In addition, the fluorescence time-resolved data pointed out that both in the absence and in the presence of glutamine the protein structure was highly rigid with small amplitude of segmental motion up to 65 degrees C and a low accessibility of the protein tryptophan residues to acrylamide. The obtained results on the structural properties of the recombinant glutamine-binding protein in the absence and in the presence of glutamine can contribute to a better understanding of the transport-related functions of the protein and structurally similar periplasmic transport proteins, as well as to the design and development of new biotechnological applications of this class of proteins.     

Rotational motion and flexibility of Ca2+,Mg2+-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes

Ca2+,Mg2+-dependent adenosine 5'-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C but increases considerably with decreasing temperature and upon fixation with glutaraldehyde. A purified Ca2+,Mg2+-dependent ATPase preparation partially depleted of membrane lipids exhibits similar properties. The low value of the anisotropy at 37 degrees C is due to the existence of a fast motion which in part is assigned to independent segmental motion of the protein. This internal flexibility of the ATPase may have considerable significance for the functional properties of the enzyme. At times longer than 20 mus, the anisotropy decays with a time constant which varies from approximately 90 mus at 0 degrees C to approximately 40 mus at 37 degrees C. This decay is assigned to rotation of the ATPase about an axis normal to the plane of the membrane. There is some evidence for self-aggregation of the protein at lower temperatures.     

Stability of aprA-subtilisin in sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.     

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

Sodium dodecyl sulfate polyacrylamide gel electrophoresis as a method for studying the stability of subtilisin

A simple method has been developed to study the stability of subtilisin. Protein incubated at various temperatures in the presence of proteinase inhibitor was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and showed a transition from the intact state to the unfolded state between 55 degrees C and 65 degrees C. Additionally, autolysis was also observed above 65 degrees C. In the absence of inhibitor, similar results were obtained below 55 degrees C; however, above 65 degrees C no protein of any size was observed due to extensive autolysis. These results demonstrate that SDS-PAGE can trap subtilisin in the state in which the protein existed prior to the analysis. It can be used to identify the different forms, including autolysis products, of the protein generated by heat denaturation. This method was used to study SDS-induced unfolding of aprA-subtilisin. When the protein was incubated with 0.25% SDS at different NaCl concentrations, a gradual increase in unfolding was observed with increasing NaCl concentration. This change paralleled a decrease in the critical micelle concentration of SDS, indicating that the rate of unfolding of aprA-subtilisin increases with increasing SDS micelle concentration. No detectable unfolding was observed below the critical micelle concentration.