Unfolding intermediates in the triple helix to coil transition of bovine type XI collagen and human type V collagens alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3

The thermal triple helix to coil transitions of two human type V collagens (alpha 1(2) alpha 2 and alpha 1 alpha 2 alpha 3) and bovine type XI collagen differ from those of the interstitial collagens type I, II, and III by the presence of unfolding intermediates. The total transition enthalpy of these collagens is comparable to the transition enthalpy of the interstitial collagens with values of 17.9 kJ/mol tripeptide units for type XI collagen, 22.9 kJ/mol for type V (alpha 1(2) alpha 2), and 18.5 kJ/mol for type V (alpha 1 alpha 2 alpha 3). It is shown by optical rotatory dispersion and differential scanning calorimetry that complex transition curves with stable intermediates exist. Type XI collagen has two main transitions at 38.5 and 41.5 degrees C and a smaller transition at 40.1 degrees C. Type V (alpha 1(2) alpha 2) shows two main transitions at 38.2 and 42.9 degrees C and two smaller transitions at 40.1 and 41.3 degrees C. Compared to these two collagens type V (alpha 1 alpha 2 alpha 3) unfolds at a lower temperature with two main transitions at 36.4 and 38.1 degrees C and two minor transitions at 40.5 and 42.9 degrees C. The intermediates present at different temperatures are characterized by resistance to trypsin digestion, length measurements of the resistant fragments after rotary shadowing, and amino-terminal sequencing. One of the intermediate peptides has been identified as belonging to the alpha 2 type V chain, starting at position 430 and being about 380 residues long. (The residue numbering begins with the first residue of the first amino-terminal tripeptide unit of the main triple helix. The alpha 2(XI) chain was assumed to be the same length as the alpha 1(XI). One intermediate was identified from the alpha 2(XI) chain and with starting position at residue 495, and three from the alpha 3(XI) with starting positions at residues 519, 585, and 618.     

Thermally induced chain exchange of frog alpha beta-tropomyosin.

The thermally induced unfolding of the alpha-helix of Rana esculenta alpha alpha, alpha beta and beta beta tropomyosin and two tryptic fragments approximately corresponding to the N- and C-terminal halves of alpha beta have been investigated by use of optical rotation, circular dichroism and UV difference spectroscopy. Reversible unfolding transitions of alpha alpha and beta beta occur around 49 degrees C and 32 degrees C, respectively. The helix unfolding of alpha beta shows two major transitions at 36 degrees C and 48 degrees C, with only the latter being reversible. The major unfolding transitions of each of the N- and C-terminal alpha beta peptides roughly correspond to the low and high temperature transitions of intact alpha beta, respectively. This suggests that the unfolding of alpha beta could be due to unfolding of two independent domains in alpha beta. UV difference data, crosslinking and chromatography results show, however, that the unfolding of alpha beta at 36 degrees C is due to chain exchange with the formation of alpha alpha homodimers and largely unfolded beta monomers, and that the transition at 48 degrees C is due to unfolding of alpha alpha dimers.     

Thermal stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

Random chemical mutagenesis, in vitro, of the 5' portion of the Escherichia coli trpA gene has yielded 66 mutant alpha subunits containing single amino acid substitutions at 49 different residue sites within the first 121 residues of the protein; this portion of the alpha subunit contains four of the eight alpha helices and three of the eight beta strands in the protein. Sixty-two of the subunits were examined for their heat stabilities by sensitivity to enzymatic inactivation (52 degrees C for 20 min) in crude extracts and by differential scanning calorimetry (DSC) with 29 purified proteins. The enzymatic activities of mutant alpha subunits that contained amino acid substitutions within the alpha and beta secondary structures were more heat labile than the wild-type alpha subunit. Alterations only in three regions, at or immediately C-terminal to the first three beta strands, were stability neutral or stability enhancing with respect to enzymatic inactivation. Enzymatic thermal inactivation appears to be correlated with the relative accessibility of the substituted residues; stability-neutral mutations are found at accessible residual sites, stability-enhancing mutations at buried sites. DSC analyses showed a similar pattern of stabilization/destabilization as indicated by inactivation studies. Tm differences from the wild-type alpha subunit varied +/- 7.6 degrees C. Eighteen mutant proteins containing alterations in helical and sheet structures had Tm's significantly lower (-1.6 to -7.5 degrees C) than the wild-type Tm (59.5 degrees C). In contrast, 6 mutant alpha subunits with alterations in the regions following beta strands 1 and 3 had increased Tm's (+1.4 to +7.6 degrees C). Because of incomplete thermal reversibilities for many of the mutant alpha subunits, most likely due to identifiable aggregated forms in the unfolded state, reliable differences in thermodynamic stability parameters are not possible. The availability of this group of mutant alpha subunits which clearly contain structural alterations should prove useful in defining the roles of certain residues or sequences in the unfolding/folding pathway for this protein when examined by urea/guaninidine denaturation kinetic analysis.     

Pressure perturbation and differential scanning calorimetric studies of bipolar tetraether liposomes derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius

Differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) were used to characterize thermal phase transitions, membrane packing, and volumetric properties in multilamellar vesicles (MLVs) composed of the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius grown at different temperatures. For PLFE MLVs derived from cells grown at 78 degrees C, the first DSC heating scan exhibits an endothermic transition at 46.7 degrees C, a small hump near 60 degrees C, and a broad exothermic transition at 78.5 degrees C, whereas the PPC scan reveals two transitions at approximately 45 degrees C and 60 degrees C. The endothermic peak at 46.7 degrees C is attributed to a lamellar-to-lamellar phase transition and has an unusually low DeltaH (3.5 kJ/mol) and DeltaV/V (0.1%) value, as compared to those for the main phase transitions of saturated diacyl monopolar diester lipids. This result may arise from the restricted trans-gauche conformational changes in the dibiphytanyl chain due to the presence of cyclopentane rings and branched methyl groups and due to the spanning of the lipid molecules over the whole membrane. The exothermic peak at 78.5 degrees C probably corresponds to a lamellar-to-cubic phase transition and exhibits a large and negative DeltaH value (-23.2 kJ/mol), which is uncommon for normal lamellar-to-cubic phospholipid phase transformations. This exothermic transition disappears in the subsequent heating scans and thus may involve a metastable phase, which is irreversible at the scan rate used. Further, there is no distinct peak in the plot of the thermal expansion coefficient alpha versus temperature near 78.5 degrees C, indicating that this lamellar-to-cubic phase transition is not accompanied by any significant volume change. For PLFE MLVs derived from cells grown at 65 degrees C, similar DSC and PPC profiles and thermal history responses were obtained. However, the lower growth temperature yields a higher DeltaV/V ( approximately 0.25%) and DeltaH (14 kJ/mol) value for the lamellar-to-lamellar phase transition measured at the same pH (2.1). A lower growth temperature also generates a less negative temperature dependence of alpha. The changes in DeltaV/V, DeltaH, and the temperature dependence of alpha can be attributed to the decrease in the number of cyclopentane rings in PLFE at the lower growth temperature. The relatively low DeltaV/V and small DeltaH involved in the phase transitions help to explain why PLFE liposomes are remarkably thermally stable and also echo the proposal that PLFE liposomes are generally rigid and tightly packed. These results help us to understand why, despite the occurrence of thermal-induced phase transitions, PLFE liposomes exhibit a remarkably low temperature sensitivity of proton permeation and dye leakage.     

Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase

Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units-does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K)-ATPase in both soluble and membranous states.     

A scanning calorimetric study of unfolding equilibria in homodimeric chicken gizzard tropomyosins.

Using both circular dichroism (CD) and differential scanning calorimetry (DSC), several laboratories find that the thermal unfolding transitions of alpha alpha and beta beta homodimeric coiled coils of rabbit tropomyosin are multistate and display an overall unfolding enthalpy of near 300 kcal (mol dimer)(-1). In contrast, an extant CD study of beta beta and gamma gamma species of chicken gizzard tropomyosin concludes that their unfolding transitions are simple two-state transitions, with much smaller overall enthalpies (98 kcal mol(-1) for beta beta and 162 kcal mol(-1) for gamma gamma). However, these smaller enthalpies have been questioned, because they imply a concentration dependence of the melting temperatures that is far larger than observed by CD. We report here DSC studies of the unfolding of both beta beta and gamma gamma chicken gizzard homodimers. The results show that these transitions are very similar to those in rabbit tropomyosins in that 1) the overall unfolding enthalpy is near 300 kcal mol(-1); 2) the overall delta C(rho) values are significantly positive; 3) the various transitions are multistate, requiring at least two and as many as four domains to fit the DSC data. DSC studies are also reported on these homodimeric species of chicken gizzard tropomyosin with a single interchain disulfide cross-link. These results are also generally similar to those for the correspondingly cross-linked rabbit tropomyosins.     

Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

Unfolding domains of recombinant fusion alpha alpha-tropomyosin.

The thermal unfolding of the coiled-coil alpha-helix of recombinant alpha alpha-tropomyosin from rat striated muscle containing an additional 80-residue peptide of influenza virus NS1 protein at the N-terminus (fusion-tropomyosin) was studied with circular dichroism and fluorescence techniques. Fusion-tropomyosin unfolded in four cooperative transitions: (1) a pretransition starting at 35 degrees C involving the middle of the molecule; (2) a major transition at 46 degrees C involving no more than 36% of the helix from the C-terminus; (3) a major transition at 56 degrees C involving about 46% of the helix from the N-terminus; and (4) a transition from the nonhelical fusion domain at about 70 degrees C. Rabbit skeletal muscle tropomyosin, which lacks the fusion peptide but has the same tropomyosin sequence, does not exhibit the 56 degrees C or 70 degrees C transition. The very stable fusion unfolding domain of fusion-tropomyosin, which appears in electron micrographs as a globular structural domain at one end of the tropomyosin rod, acts as a cross-link to stabilize the adjacent N-terminal domain. The least stable middle of the molecule, when unfolded, acts as a boundary to allow the independent unfolding of the C-terminal domain at 46 degrees C from the stabilized N-terminal unfolding domain at 56 degrees C. Thus, strong localized interchain interactions in coiled-coil molecules can increase the stability of neighboring domains.     

Effect of the chirality of the glycerol backbone on the bilayer and nonbilayer phase transitions in the diastereomers of di-dodecyl-beta-D-glucopyranosyl glycerol.

We have studied the physical properties of aqueous dispersions of 1,2-sn- and 2,3-sn-didodecyl-beta-D-glucopyranosyl glycerols, as well as their diastereomeric mixture, using differential scanning calorimetry and low angle x-ray diffraction. Upon heating, both the chiral lipids and the diastereomeric mixture exhibit characteristically energetic L beta/L alpha phase transitions at 31.7-32.8 degrees C and two or three weakly energetic thermal events between 49 degrees C and 89 degrees C. In the diastereomeric mixture and the 1,2-sn glycerol derivative, these higher temperature endotherms correspond to the formation of, and interconversions between, several nonlamellar structures and have been assigned to L alpha/QIIa, QIIa/QIIb, and QIIb/HII phase transitions, respectively. The cubic phases QIIa and QIIb, whose cell lattice parameters are strongly temperature dependent, can be identified as belonging to space groups Ia3d and Pn3m/Pn3, respectively. In the equivalent 2,3-sn glucolipid, the QIIa phase is not observed and only two transitions are seen at 49 degrees C and 77 degrees C, which are identified as L alpha/QIIb and QIIb/HII phase transitions, respectively. These phase transitions temperatures are some 10 degrees C lower than those of the corresponding phase transitions observed in the diastereomeric mixture and the 1,2-sn glycerol derivative. On cooling, all three lipids exhibit a minor higher temperature exothermic event, which can be assigned to a HII/QIIb phase transition. An exothermic L alpha/L beta phase transition is observed at 30-31 degrees C. A shoulder is sometimes discernible on the high temperature side of the L alpha/L beta event, which may originate from a QIIb/L alpha phase transition prior to the freezing of the hydrocarbon chains. None of the lipids show evidence of a QIIa phase on cooling. No additional exothermic transitions are observed on further cooling to -3 degrees C. However, after nucleation at 0 degrees C followed by a short period of annealing at 22 degrees C, the 1,2-sn glucolipid forms an Lc phase that converts to an L alpha phase at 39.5 degrees C on heating. Neither the diastereomeric mixture nor the 2,3-sn glycerol derivative shows such behavior even after extended periods of annealing. Our results suggest that the differences in the phase behavior of these glycolipid isomers may not be attributable to headgroup size per se, but rather to differences in the stereochemistry of the lipid polar/apolar interfacial region, which consequently effects hydrogen-bonding, hydration, and the hydrophilic/hydrophobic balance.     

Thermal stability of myosin rod from various species

The radius of gyration and fraction helix as a function of temperature have been determined for myosin rod from four different species: rabbit, frog, scallop, and antarctic fish. Measurements from sodium dodecyl sulfate gel electrophoresis indicate that all particles have the same molecular weight (approximately 130K). All fragments are nearly 100% alpha-helical at low temperatures (0-5 degrees C). The melting profiles for each are qualitatively similar in shape, but their midpoints are shifted along the temperature axis in the following order: antarctic fish (Tm = 33 degrees C), scallop (Tm = 39 degrees C), frog (Tm = 45 degrees C), and rabbit (Tm = 49 degrees C). Corresponding radius of gyration vs temperature profiles for each species are shifted to lower temperatures (approximately 5-8 degrees C) with respect to the optical rotation melting curves. From plots of radius of gyration vs fraction helix, we find a marked drop in the radius of gyration (from 43 to approximately 34 nm) with less than a 5% decrease in fraction helix for rabbit, frog, and antarctic fish rods, whereas the radius of gyration of scallop rod never exceeds 34 nm. Results indicate hinging of the myosin rod of each species. The thermal stabilities of the myosin rods shift in parallel with the working temperature of their respective muscles.     

Effect of single amino acid substitutions at positions 49 and 60 on the thermal unfolding of the tryptophan synthase alpha subunit from Salmonella typhimurium

We have used circular dichroism measurements to compare the thermal unfolding of the wild type tryptophan synthase alpha subunit from Salmonella typhimurium with that of seven mutant forms with single amino acid replacements at two active site residues. Glutamic acid 49 has been replaced by phenylalanine, glutamine, or aspartic acid. Aspartic acid 60 has been replaced by alanine, aspartic acid, asparagine, or tyrosine. Thermodynamic properties (delta G, delta H, delta S, and Tm) of the wild type and mutant forms have been determined experimentally by measuring the free energy of unfolding as a function of temperature. Increasing the pH from 7.0 to 8.8 decreases the tm of the wild type alpha subunit from 56 to 45 degrees C. The thermal unfolding of the wild type alpha subunit and of six of the seven mutant forms can be described as reversible, two-state transitions. In contrast, the melting curve of a mutant alpha subunit in which aspartic acid 60 is replaced by tyrosine indicates the presence of a folding intermediate which may correspond to a "molten globule." Correlations between our observations and previous folding studies and the X-ray crystallographic structure are presented. Substitution of glutamic acid 49, which is located in the hydrophobic "pit" of an eight-fold alpha/beta barrel, by a hydrophobic phenylalanine residue increases the tm from 56 to 60 degrees C. In contrast, replacement of aspartic acid 60, which is accessible to solvent, results in small reductions in the thermal stability.     

Temperature and adrenoceptors in the frog heart

1. Cardiac adrenergic receptors in a frog, Rana tigrina, were examined in winter and summer months using isolated atria preparation maintained at 24 degrees, 14 degrees and 6 degrees C. Treatments included an examination of the atrial responses to selective alpha and beta adrenergic agonists (phenylephrine and isoproterenol respectively) and antagonists (phentolamine and propranolol). 2. Basal atrial beating rates differed between summer and winter months and increased with temperature. 3. Phenylephrine produced dose-dependent increases in the atrial beating rate and tension in the winter frogs only at 6 degrees C. These increases were blunted by phentolamine. 4. Isoproterenol produced positive chronotropic effects of 14 degrees and 24 degrees C but not at 6 degrees C in both summer and winter frogs; these effects were abolished by propranolol. Further, at 6 degrees C, the contractile response of the atrial tissue to isoproterenol was very sensitive. 5. Data suggests that the alpha adrenoceptor might be physiologically important to the frog in the low temperature environment of the cold season, during which period the cardiac beta adrenergic activity would be minimal or even absent.     

Bilayer properties of totally synthetic C16:0-lactosyl-ceramide

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the structural and thermal properties of totally synthetic D-erythro-N-palmitoyl-lactosyl-C(18)-sphingosine (C16:0-LacCer). Over the temperature range 0-90 degrees C, fully hydrated C16:0-LacCer shows complex thermal transitions characteristic of polymorphic behavior of exclusively bilayer phases. On heating at 5 degrees C/min, hydrated C16:0-LacCer undergoes a complex two-peak endothermic transition with maxima at 69 degrees C and 74 degrees C and a total enthalpy of 14.6 kcal/mol C16:0-LacCer. At a slower heating rate (1.5 degrees C/min), two endothermic transitions are observed at 66 degrees C and 78 degrees C. After cooling to 0 degrees C, the subsequent heating run shows three overlapping endothermic transitions at 66 degrees C, 69 degrees C, and 71.5 degrees C, followed by a chain-melting endothermic transition at 78 degrees C. Two thermal protocols were used to completely convert C16:0-LacCer to its stable, high melting temperature (78 degrees C) form. As revealed by x-ray diffraction, over the temperature range 20-78 degrees C this stable phase exhibits a bilayer structure, periodicity d approximately 65 A with an ordered chain packing mode. At the phase transition (78 degrees C) chain melting occurs, and C16:0-LacCer converts to a liquid crystalline bilayer (L(alpha)) phase of reduced periodicity d approximately 59 A. On cooling from the L(alpha) phase, C16:0-LacCer converts to metastable bilayer phases undergoing transitions at 66-72 degrees C. These studies allow comparisons to be made with the behavior of the corresponding C16:0-Cer (. J. Lipid Res. 36:1936-1944) and C16:0-GluCer and C16:0-GalCer (. J. Lipid Res. 40:839-849). Our systematic studies are aimed at understanding the role of oligosaccharide complexity in regulating glycosphingolipid structure and properties.     

Conformational and hydrational properties during the L(beta)- to L(alpha)- and L(alpha)- to H(II)-phase transition in phosphatidylethanolamine

Differential scanning calorimetry (DSC) measurements have been carried out simultaneously with small- and wide-angle X-ray scattering recordings on liposomal dispersions of stearoyl-oleoyl-phosphatidylethanolamine (PE) in a temperature range from 20 to 80 degrees C. The main transition temperature, T(m), was determined at 30.9 degrees C with an enthalpy of 28.5 kJ/mol and the lamellar-to-inverse hexagonal phase transition temperature, T(hex), at 61.6 degrees C with an enthalpy of 3.8 kJ/mol. Additionally highly resolved small angle X-ray diffraction experiments performed at equilibrium conditions allowed a reliable decomposition of the lattice spacings into hydrophobic and hydrophilic structure elements as well as the determination of the lipid interface area of the lamellar gel-phase (L(beta)), the fluid lamellar phase (L(alpha)) and of the inverse hexagonal phase (H(II)). The rearrangement of the lipid matrix and the coincident change of free water per lipid is illustrated for both transitions. Last, possible transition mechanisms are discussed on a molecular level.     

Inhibition of cell surface receptor-bound plasmin by alpha 2-antiplasmin and alpha 2-macroglobulin.

The purpose of this investigation was to characterize the reaction of alpha 2-antiplasmin (alpha 2AP) and alpha 2-macroglobulin (alpha 2M) with human plasmin bound to rat C6 glioma cells and human umbilical vein endothelial cells (HUVECs). Binding of plasmin (0.1 microM) to C6 cells at 4 degrees C did not cause cell detachment, decrease viability or change cell morphology. The KD and Bmax for the binding of diisopropyl phosphoryl plasmin (DIP-plasmin) to C6 cells were 0.9 microM and 2.6 x 10(6) sites/cell. The dissociation rate constants (koff) for 125I-plasmin were 9.7 x 10(-4) and 4.0 x 10(-4) s-1 at 4 degrees C in the presence and absence of 0.3 microM DIP-plasmin, respectively. Similar constants were determined for 125I-plasminogen and 125I-DIP-plasmin. Neither alpha 2AP nor alpha 2M affected the dissociation of DIP-plasmin. C6 cell-associated 125I-plasmin reacted slowly with alpha 2AP; however, the inhibition rate constants exceeded the koff. alpha 2AP-plasmin complex formed after the plasmin dissociated into solution (reaction pathway 1) and by direct reaction of alpha 2AP with cell-associated enzyme (reaction pathway 2). High concentrations of alpha 2AP favored pathway 2. C6 cell-associated plasmin was also protected from inhibition by alpha 2M. While the same pathways were probably involved in this reaction, alpha 2M was less effective than alpha 2AP as an inhibitor of nondissociated plasmin (pathway 2). When C6 cell-bound plasmin reacted with alpha 2AP, alpha 2AP-plasmin complex was recovered primarily in the medium, suggesting dissociation of complexes formed on the cell surface. Plasmin-receptor dissociation and inhibition experiments were performed at 22 degrees and 37 degrees C, confirming the conclusions of the 4 degrees C studies. Comparable results were also obtained using HUVEC cultures. These studies demonstrate that cell-associated plasmin is protected from inhibition by alpha 2M as well as alpha 2AP. At least two reaction pathways may be demonstrated for the inhibition of plasmin that is initially receptor-bound; however, neither pathway is highly effective, accounting for the "plasmin-protective" activity of the cell surface.     

Co-solvent effects on structure and function properties of savinase: solvent-induced thermal stabilization

The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70 degrees C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme's activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70 degrees C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives.     

Isolation and physico-chemical properties of a protein, included in an endotoxin from Yersinia pseudotuberculosis

A major protein of the endotoxin from Yersinia pseudotuberculosis was isolated from the complex lipid A--protein by treatment with SDS and triton X-100 followed by gel-chromatography on Sephacryl S-300. Protein has apparent molecular mass 40 kDa and alanine as N-terminal amino acid residue. CD and IR spectroscopy conformational changes of the protein molecule in the process of its isolation. The thermal and pH stabilities of the protein were investigated by the methods of intrinsic fluorescence and differential scanning microcalorimetry. The isolated protein revealed two thermal transitions (at 30-35 and 50-55 degrees C), which depend on Ca2+ concentration.     

Differences in thermal stability between reduced and oxidized cytochrome b562 from Escherichia coli

The thermal stabilities of ferri- and ferrocytochrome b562 were examined. Thermally induced spectral changes, monitored by absorption and second-derivative spectroscopies, followed the dissociation of the heme moiety and the increased solvation of tyrosine residue(s) located in close proximity to the heme binding site. All observed thermal transitions were independent of the rate of temperature increase (0.5-2 degrees C/min), and the denatured protein exhibited partial to near-complete reversibility upon return to ambient temperature. The extent of renaturation of cytochrome b562 is dependent on the amount of time the unfolded conformer is exposed to temperatures above the transition temperature, Tm. All thermally induced spectra changes fit a simple two-state model, and the thermal transition was assumed to be reversible. The thermal transition for ferrocytochrome b562 yielded Tm and van't Hoff enthalpy (delta HvH) values of 81.0 degrees C and 137 kcal/mol, respectively. In contrast, Tm and delta HvH values obtained for the ferricytochrome were 66.7 degrees C and 110 kcal/mol, respectively. The estimated increase in the stabilization free energy at the Tm of ferricytochrome b562 following the one-electron reduction to the ferrous form, where delta delta G = delta Tm delta Sm [delta Sm = 324 cal/(K.mol), delta Tm = 14.3 degrees C] [Becktel, W. J., & Schellman, J. A. (1987) Biopolymers 26, 1859-1877], is 4.6 kcal/mol.     

De- and rehydration behavior of alpha,alpha-trehalose dihydrate under humidity-controlled atmospheres

Effects of humidity were investigated on de- and rehydration behavior of alpha,alpha-trehalose dihydrate (T(h)) throughout simultaneous measurements of differential scanning calorimetry and X-ray diffractometry (DSC-XRD) and simultaneous thermogravimetry and differential thermal analysis (TG-DTA). When T(h) was heated from room temperature under dry nitrogen atmosphere, a metastable anhydrous crystal (T(alpha)) was formed at 105 degrees C after dehydration of T(h). The resulting T(alpha) melted at 125 degrees C and became amorphous, followed by cold crystallization from 150 degrees C giving rise to a stable anhydrous crystal T(beta). Under a highly humid atmosphere, on the other hand, T(beta) was formed at 90 degrees C directly as a result of T(h) dehydration. T(alpha) was readily rehydrated and turned back to T(h) when nitrogen gas with low water vapor pressure of 2.1kPa was admitted, whereas high water vapor pressure up to 7.4kPa was required for rehydration of T(beta) into T(h). This study provided a picture of pathways that link various solid forms of trehalose, taking into account the effects of a humid environment.     

Effect of temperature and low pH on structure and stability of matrix porin in micellar detergent solutions

Thermal and low pH stabilities of matrix porin (Omp F) solubilized in the micellar solutions of ionic (SDS) and nonionic detergents were investigated by the methods of circular dichroism, intrinsic fluorescence, light scattering and sedimentation velocity. The stability of porin structure in solution is much higher in the presence of beta-octyl glucoside than with SDS. In the presence of SDS, sharp transitions were detected by all parameters measured, above 55 degrees C at neutral pH and below pH 4.5 at 20 degrees C. These transitions involve at least three concomitant processes: unfolding of protein, dissociation of trimers to monomers and the disruption of the protein-detergent micellar complexes, all events being irreversible in the presence of SDS. The nonionic detergent, beta-octyl glucoside, increases the stability of porin in acidic conditions, since neither dissociation nor denaturation was observed in the pH region between 7.5 and 2.0. However, at pH less than 3.5, small, reversible changes in protein structure became evident. The thermal stability of porin is also increased by beta-octyl glucoside as evidenced by a transition temperature 15-20 degrees C higher as compared to SDS. A considerable degree of native porin structure was regained after heat treatment in the presence of beta-octyl glucoside, though the reconstituted trimers were not identical to the native ones. The addition of lipopolysaccharide and divalent cations (Ca2+, Mg2+) to the experimental system did not improve the thermal reversibility.