Thermodynamic analysis of denaturant-induced unfolding of HodC69S protein supports a three-state mechanism.

Thermodynamic stability parameters and the equilibrium unfolding mechanism of His 6HodC69S, a mutant of 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) having a Cys to Ser exchange at position 69 and an N-terminal hexahistidine tag (His 6HodC69S), have been derived from isothermal unfolding studies using guanidine hydrochloride (GdnHCl) or urea as denaturants. The conformational changes were monitored by following changes in circular dichroism (CD), fluorescence, and dynamic light scattering (DLS), and the resulting transition curves were analyzed on the basis of a sequential three-state model N = I = D. The structural changes have been correlated to catalytic activity, and the contribution to stability of the disulfide bond between residues C37 and C184 in the native protein has been established. A prominent result of the present study is the finding that, independent of the method used for denaturing the protein, the unfolding mechanism always comprises three states which can be characterized by, within error limits, identical sets of thermodynamic parameters. Apparent deviations from three-state unfolding can be rationalized by the inability of a spectroscopic probe to discriminate clearly between native, intermediate, and unfolded ensembles. This was the case for the CD-monitored urea unfolding curve.

Defining thresholds of sustainable impact on benthic communities in relation to fishing disturbance.

While the direct physical impact on seabed biota is well understood, no studies have defined thresholds to inform an ecosystem-based approach to managing fishing impacts. We addressed this knowledge gap using a large-scale experiment that created a controlled gradient of fishing intensity and assessed the immediate impacts and short-term recovery. We observed a mosaic of taxon-specific responses at various thresholds. The lowest threshold of significant lasting impact occurred between 1 and 3 times fished and elicited a decrease in abundance of 39 to 70% for some sessile epifaunal organisms (cnidarians, bryozoans). This contrasted with significant increases in abundance and/or biomass of scavenging species (epifaunal echinoderms, infaunal crustaceans) by two to four-fold in areas fished twice and more. In spite of these significant specific responses, the benthic community structure, biomass and abundance at the population level appeared resilient to fishing. Overall, natural temporal variation in community metrics exceeded the effects of fishing in this highly dynamic study site, suggesting that an acute level of disturbance (fished over six times) would match the level of natural variation. We discuss the implications of our findings for natural resources management with respect to context-specific human disturbance and provide guidance for best fishing practices.

Pharmacokinetics of the in vivo and in vitro conversion of 9-nitro-20(S)-camptothecin to 9-amino-20(S)-camptothecin in humans, dogs, and mice.

We have determined that 9-nitro-20(S)-camptothecin (9NC) converts to 9-amino-20(S)-camptothecin (9AC) in humans, dogs, and mice. Following a single oral dose of 0.1 mg/kg of 9NC, the human plasma concentration reached a maximum concentration of 483 ng/ml at 3.4 h with an area under the curve (AUC) of 2.6 micrograms.h/ml and a half-life of 2.5 h. As conversion of 9NC to 9AC occurred, the maximum calculated concentration of 9AC was 14.0 ng/ml at 10.3 h with an AUC of 311 ng.h/ml and a half-life of 7.1 h. Following a single oral dose of 1.0 mg/kg of 9NC, the maximum concentration of 9NC in the human volunteer was 1247 ng/ml at 5.3 h with an AUC of 17194 ng.h/ml and a half-life of 4.9 h. In this human, the Cmax of 9AC was 208 ng/ml at 17.2 h; the AUC was determined to be 9121 ng.h/ml, and the half-life was 13.1 h. In a dog after a single oral dose of 1.0 mg/kg 9NC, the maximum concentration for 9NC was 19.1 ng/ml at 0.7 h with a half-life of 6.4 h and an AUC of 186 ng.h/ml. The maximum concentration of 9AC in this dog was 9.2 ng/ml at 2.9 h with an AUC of 310 ng.h/ml and a half-life of 21.1 h. The maximum concentration of 9NC in the mouse after a single oral dose of 4.1 mg/kg of 9NC was 732 ng/ml at time 0.1 h with an AUC of 441 ng.h/ml and a half-life of 10.0 h. The maximum concentration of 9AC in the mouse was 26 ng/ml at 0.6 h. The AUC was 63 ng.h/ml, and the half-life was 1.2 h. Incubation of mouse liver, spleen, kidney, brain, and muscle tissue with 9NC all indicated conversion to 9AC, yet no conversion was observable in cell-free plasma from human or mouse blood. Structural identification of 9AC was confirmed by mass spectrometry.

Camptothecin and its derivatives induce expression of the c-jun protooncogene in human myeloid leukemia cells.

We have recently demonstrated that certain camptothecin derivatives are effective agents in the treatment of human tumor xenografts in nude mice. While camptothecin and its derivatives are recognized as inhibitors of topoisomerase I, little is known about the effects of these agents on specific gene expression, particularly genes involved in growth control. The c-jun early response gene codes for a leucine zipper transcription factor. The present studies demonstrate that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin inhibit the growth of human U-937 myeloid leukemia cells and induce expression of the c-jun gene. c-jun transcripts were increased at 3 h and reached a maximum at 6 h of drug exposure. We also demonstrate that the induction of c-jun gene expression by these agents occurs at the transcriptional level. H7, a nonselective inhibitor of protein kinase C, completely blocked c-jun expression in 20(S)-camptothecin-treated cells, while another protein kinase inhibitor, HA1004, had no detectable effect. Similar findings were obtained for other leucine zipper encoding genes, including jun-B. These results suggest that 20(S)-camptothecin, 9-amino-20(S)-camptothecin, and 9-nitro-20(S)-camptothecin activate a cellular response involving the induction of early response genes. Finally, we demonstrate that induction of c-jun expression occurs in association with internucleosomal DNA fragmentation, a characteristic of programmed cell death.

Complete growth inhibition of human cancer xenografts in nude mice by treatment with 20-(S)-camptothecin.

20-(S)-Camptothecin (CAM), a plant alkaloid, was tested against 13 human cancer xenograft lines carried by immunodeficient (nude) mice. The drug, formulated in 20% intralipid and given i.m., was more effective than any other clinically available drug tested. It was found that: (a) CAM, at nontoxic doses, suppressed growth and induced regression of cancer of the colon (3 lines), lung (4 lines), breast (2 lines), stomach (1 line), ovary (1 line), and malignant melanoma (2 lines); (b) the drug was equally effective administered i.m. or p.o. Both routes are significantly better than i.v. administration; (c) CAM is substantially more effective and less toxic than its sodium salt, which was unsuccessfully tested in cancer patients. CAM should be further tested against responsive cancers as a drug which is easy to isolate and formulate for large-scale studies.

Regression of human breast carcinoma tumors in immunodeficient mice treated with 9-nitrocamptothecin: differential response of nontumorigenic and tumorigenic human breast cells in vitro.

We have shown recently that the plant alkaloid camptothecin and its derivatives inhibited growth of human carcinoma and melanoma cells in vitro and induced regression of advanced human malignant melanoma tumors growing in immunodeficient (nude) mice. Here, we have extended these studies to show that the camptothecin derivative 9-nitro-20(S)- camptothecin (9NC) induces complete regression of advanced breast carcinoma tumors growing in nude mice. We also report that 9NC inhibits growth of nontumorigenic and tumorigenic breast cells in vitro. However, flow cytometry studies show that 9NC elicits differential effects on the cell cycle of nontumorigenic and tumorigenic cells. In general, 9NC-treated nontumorigenic cells accumulate slowly at the G2 phase of the cell cycle with no cell death. In contrast, 9NC-treated tumorigenic cells transverse rapidly from G1 to S phase followed by cell death. Removal of 9NC from the cell cultures resulted in most nontumorigenic cells dividing, whereas tumorigenic cells continued to die after removal of 9NC. Taken together, the findings indicate a different response of nontumorigenic and tumorigenic breast cells to 9NC.

Complete inhibition of growth followed by death of human malignant melanoma cells in vitro and regression of human melanoma xenografts in immunodeficient mice induced by camptothecins.

The plant alkaloid camptothecin and three camptothecin derivatives were used to study responses of human malignant melanoma (BRO) cells xenografted in immunodeficient (nude) mice. Camptothecin and its derivatives 9-nitro-20(S)-camptothecin and 9-amino-20(S)-camptothecin inhibited growth of tumors and caused regression in all tumor-bearing mice. Tumor regression was accompanied by degenerative changes in the tumor cells, as assessed by microscopic observations of histological sections prepared from the tumors. No toxic effects were observed in the drug-treated mice, with or without xenografts. In parallel experiments, camptothecin, 9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin inhibited proliferation of BRO cells in vitro and resulted in dramatic morphological cellular changes comparable to those observed in the sections of solid tumors. The derivative 12-nitro-20(S)-camptothecin had no effect on BRO tumors or cell cultures. The difference between 9-nitro-20(S)-camptothecin and 12-nitro-20(S)-camptothecin is the position at which the NO2 group is attached to the camptothecin molecule. In contrast to BRO melanoma cells, none of the camptothecin derivatives had any effect on cultured human melanocytes, the normal counterparts of melanoma cells. Taken together, the findings indicate that camptothecin and derivatives exert different effects on the growth and morphology of normal and malignant (BRO) melanocytes.

A new alternative method to quantify residual structure in 'unfolded' proteins.

Pig (pCSD1) and human (hCSD1) calpastatin domain 1 proteins were studied to characterize common features of the denatured state of proteins. These proteins were chosen for the present investigation, because pCSD1 was suggested previously to be unstructured in water even at 25 degrees C (1) [T. Konno et al., Biochim. Biophys. Acta 1342 (1997) 73-82]. hCSD1 could be expected to exhibit similar features on the basis of preliminary spectroscopic studies. In the present study, the experimental grounds for the estimate of residual structure in the unfolded state were differential scanning calorimetry heat capacity and circular dichroism (CD) measurements over the temperature range 10-80 degrees C. At selected temperatures, we studied also the effect of guanidinium hydrochloride (GdnHCl) which is known to promote further unfolding of the polypeptide chain. All other measurements were performed at pH 6 in pure water. The present results support the conclusion that the comparison of the experimentally obtained heat capacity data with theoretical heat capacity values calculated on the basis of a newly established increment system gives insight into the degree of hydration of the unfolded polypeptide chain. The percentage by which the experimental heat capacity of the unfolded polypeptide chain differs from the calculated heat capacity permits a quantitative estimate of the residual structure. This estimate is in good agreement with that based on CD absorption. The heat capacity approach has the advantage of comparing fully hydrated and partially hydrated residues in the same aqueous environment, whereas for example spectroscopic measurements, such as CD, are generally referred to the fully unfolded chain in concentrated urea or GdnHCl solutions. As the unfolded chains of pCSD1 and hCSD1 exhibit a smaller heat capacity than that calculated on the new peptide-based increment system [M. Häckel et al., J. Mol. Biol. 291 (1999) 197-213], we conclude that the residues in the unfolded polypeptide chain are less hydrated than the same residues in oligopeptides. This suboptimal hydration is the result of residual structure in the chain as observed in both CD and heat capacity measurements.

Stability and binding properties of wild-type and c17s mutated human sterol carrier protein 2.

The temperature- and solvent-induced denaturation of both the SCP2 wild-type and the mutated protein c71s were studied by CD measurements at 222 nm. The temperature-induced transition curves were deconvoluted according to a two-state mechanism resulting in a transition temperature of 70.5 degrees C and 59.9 degrees C for the wild-type and the c71s, respectively, with corresponding values of the van't Hoff enthalpies of 183 and 164 kJ/mol. Stability parameters characterizing the guanidine hydrochloride denaturation curves were also calculated on the basis of a two-state transition. The transitions of the wild-type occurs at 0.82 M GdnHCl and that of the c71s mutant at 0.55 M GdnHCl. These differences in the half denaturation concentration of GdnHCl reflect already the significant stability differences between the two proteins. A quantitative measure are the Gibbs energies DeltaG(0)(D)(buffer) at 25 degrees C of 15.5 kJ/mol for the wild-type and 8.0 kJ/mol for the mutant. We characterized also the alkyl chain binding properties of the two proteins by measuring the interaction parameters for the complex formation with 1-O-Decanyl-beta-D-glucoside using isothermal titration microcalorimetry. The dissociation constants, K(d), for wild-type SCP2 are 335 microM at 25 degrees C and 1.3 mM at 35 degrees C. The corresponding binding enthalpies, DeltaH(b), are -21. 5 kJ/mol at 25 degrees C and 72.2 kJ/mol at 35 degrees C. The parameters for the c71s mutant at 25 degrees C are K(d)=413 microM and DeltaH(b)=16.6 kJ/mol. These results suggest that both SCP2 wild-type and the c71s mutant bind the hydrophobic compound with moderate affinity.

Folding energetics of ligand binding proteins. I. Theoretical model.

Heat capacity curves as obtained from differential scanning calorimetry are an outstanding source for molecular information on protein folding and ligand-binding energetics. However, deconvolution of C(p) data of proteins in the presence of ligands can be compromised by indeterminacies concerning the correct choice of the statistical thermodynamic ensemble. By convent, the assumption of constant free ligand concentration has been used to derive formulae for the enthalpy. Unless the ligand occurs at large excess, this assumption is incorrect. Still the relevant ensemble is the grand canonical ensemble. We derive formulae for both constraints, constancy of total or free ligand concentration and illustrate the equations by application to the typical equilibrium Nx <=> N + x <=> D + x. It is demonstrated that as long as the thermodynamic properties of the ligand can be completely corrected for by performing a reference measurement, the grand canonical approach provides the proper and mathematically significantly simpler choice. We demonstrate on the two cases of sequential or independent ligand-binding the fact, that similar binding mechanisms result in different and distinguishable heat capacity equations. Finally, we propose adequate strategies for DSC experiments as well as for obtaining first estimates of the characteristic thermodynamic parameters, which can be used as starting values in a global fit of DSC data.

Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters.

The stability parameters delta Gst, delta Hst and delta Sst of native basic pancreatic trypsin inhibitor (BPTI) have been characterized by microcalorimetric unfolding studies in various buffer solutions, at different pH values and in the presence of guanidine hydrochloride. The unfolding enthalpy of BPTI, in contrast ot other globular proteins, exhibits a very small dependence on temperature, which results in a characteristic different temperature dependence of the Gibbs energy of stabilization. BPTI has a very high specific Gibbs energy of stabilization, which renders the slow exchange rates of amide protons understandable. Comparison of the unfolding entropy of BPTI at 110 degrees C with corresponding values of other proteins, revealed that the delta S values of BPTI are lower by 2.9 J/(K X residue). This lower value of the unfolding entropy is in good agreement with predictions of a theoretical study by Poland & Scheraga (1965) where the influence of crosslinks on the configurational entropy has been studied. Additionally, we were able to calculate an interaction enthalpy per site of -5.6 kJ/mol based on the measurements of unfolding of BPTI in 6 M-guanidine hydrochloride.

A new set of peptide-based group heat capacities for use in protein stability calculations.

The partial molar heat capacities of the tripeptides of the sequence glycyl-X-glycine, where X is one of the amino acids leucine, threonine, glutamine, phenylalanine, histidine, cysteine, proline, glutamic acid or arginine, and of the two tetrapeptides tetraglycine and glycyltryptophanylglycylglycine in aqueous solution over the temperature range 10-100 degrees C have been determined using high sensitivity scanning microcalorimetry. These results were used to derive the partial molar heat capacities of the various amino acid side-chains. This report completes our programme to derive reliable side-chain heat capacities for all 20 amino acids of proteins over a wide temperature range using the tripeptides Gly-X-Gly as realistic model compounds. Included in the study is a summary of the partial molar heat capacities of all 20 amino acid side-chains. These results, along with the heat capacity of the peptide backbone group, were used to calculate the partial molar heat capacities of some oligopeptides and of the random coil form of some unfolded proteins in water. The calculated heat capacities of the proteins obtained using this new set of heat capacities for the constituent groups are consistent with the heat capacities of the denatured state determined experimentally.

Thermodynamics of unfolding of the alpha-amylase inhibitor tendamistat. Correlations between accessible surface area and heat capacity.

Unfolding of the small alpha-amylase inhibitor tendamistat (74 residues, 2 disulfide bridges) has been characterized thermodynamically by high sensitivity scanning microcalorimetry. To link the stability parameters with structural information we use heat capacity group parameters and water accessible surface areas to calculate the change in heat capacity on unfolding of tendamistat. Our results show that both the group parameter and surface area approaches provide a reasonable, though not perfect, basis for delta Cp calculations. When using the experimentally determined temperature-independent heat capacity increase of 2.89 kJ mol-1 K-1 tendamistat exhibits convergence of thermodynamic parameters at about 140 degrees C, in agreement with recent predictions of the temperature at which the hydrophobic hydration is supposed to disappear. Despite the apparent support of this new view of the hydrophobic effect, there are inconsistencies in the interpretation of the thermodynamic parameters and these are addressed in the Discussion. The specific stability of tendamistat is similar to that of modified bovine pancreatic trypsin inhibitor, with only two of the native three disulfide bridges intact. This observation confirms our previous conclusion that disulfide bridges affect significantly the enthalpy and entropy of unfolding. The recent study by Doig & Williams provides additional convincing support for this conclusion. The predictive scheme proposed by these authors permits a fair estimate of the Gibbs free energy and enthalpy changes of these two proteins.

Folding energetics of ligand binding proteins II. Cooperative binding of Ca2+ to annexin I.

The calcium binding properties of annexin I as observed by thermodynamic DSC studies have been compared to the structural information obtained from X-ray investigation. The calorimetric experiment permitted to evaluate both the reaction scheme - including binding of ligand and conformational changes - and the energetics of each reaction step. According to published X-ray data Annexin I has six calcium binding sites, three medium-affinity type II and three low-affinity type III sites. The present study shows that at 37 degrees C annexin I binds in a Hill type fashion simultaneously two calcium ions in a first step with medium affinity at a concentration of 0.6 mM and another three Ca(2+) ions again cooperatively at 30 mM with low affinity. Therefore it can be concluded that only two medium-affinity type II binding sites are available. The third site, that should be accessible in principle appears to be masked presumably due to the presence of the N terminus. In view of the large calcium concentration needed for saturation of the binding sites, annexin I may be expected to be Ca(2+) free in vivo unless other processes such as membrane interaction occur simultaneously. This assumption is consistent with the finding, that the affinity of annexins to calcium is usually markedly increased by the presence of lipids.

A comparison of the energetics of annexin I and annexin V.

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.

Mechanism of protein stabilization by disulfide bridges: calorimetric unfolding studies on disulfide-deficient mutants of the alpha-amylase inhibitor tendamistat.

The present differential scanning calorimetry and circular dichroism studies on the mechanism of protein stabilization by disulfide bonds were concerned with two questions: is the increase in unfolding entropy upon removal of disulfide links sufficient for the explantation of the general stability decrease of disulfide-deficient mutants? Is it immaterial by which residue cysteine residues are replaced when disulfide bridges are to be opened? To answer these questions we investigated two disulfide bridge mutants of the alpha-amylase inhibitor Tendamistat where the large loop (C45A/C73A) or the small loop (C11A/C27A) had been opened by recombinant DNA techniques, and we compared the stability of the mutated proteins with that of wild-type Tendamistat published previously. To elucidate the significance of the nature of the group that replaces Cys we introduced in position 27 of the small loop four different amino acids instead of Cys: Ala, Leu, Ser and Thr. Surprisingly, opening of the small loop (17 residues) causes larger destabilization than opening of the large loop comprising 29 residues. The thermodynamic parameters at pH 7.0 are: wild-type: t1/2 = 81.6 degrees C, delta Hcal = 296 kJ mol-1, large loop mutant (C45A/C73A): t1/2 = 58.6 degrees C, delta Hcal = 225 kJ mol-1 and small loop mutant (C11A/C27A): t1/2 = 42.7 degrees C, delta Hcal = 135 kJ mol-1. This finding is at variance with the entropy hypothesis. The relative contributions to stability of enthalpic and entropic terms can be varied by a proper choice of substitutions. While the destabilization originating from C45A/C73A exchanges in the large loop turns out to be purely entropic, the stability decreases of the small loop mutants are caused by changes in both enthalpic and entropic terms. Leu or Ser in position 27 leads to an overall enthalpic destabilization. Thr in position 27 increases the transition enthalpy of this mutant to the value of the wild-type protein but increases at the same time the value of the transition entropy with the result of an overall entropic destabilization. Finally, in the C11A/C27A small loop mutant of lowest stability a very large enthalpic destabilization occurs, which is, however, partly counterbalanced by a reduction in the transition entropy. The preferential perturbation of the native state by the mutations is manifest in the increase of the native state heat capacity relative to that of the wild-type protein and the identity of the heat capacity of the unfolded state.(ABSTRACT TRUNCATED AT 400 WORDS)

Dimer-to-tetramer transformation: loop excision dramatically alters structure and stability of the ROP four alpha-helix bundle protein.

The ROP loop excision mutant RM6 shows dramatic changes in structure and stability in comparison to the wild-type protein. Removal of the five amino acids (Asp30, Ala31, Asp32, Glu33, Gln34) from the loop results in a complete reorganization of the protein as evidenced by single crystal X-ray analysis and thermodynamic unfolding studies. The homodimeric four-alpha-helix motif of the wild-type structure is given up. Instead a homotetrameric four-alpha-helix structure with extended, loop-free helical monomers is formed. This intriguing structural change is associated with the acquisition of hyperthermophilic stability. This is evident in the shift in transition temperature from 71 degreesC characteristic of the wild-type protein to 101 degreesC for RM6. Accordingly the Gibbs energy of unfolding is increased from 71.7 kJ (mol of dimer)-1 to 195.1 kJ (mol of tetramer)-1. The tetramer-to-monomer transition proceeds highly cooperatively involving an enthalpy change of DeltaH=1073+/-30 kJ (mol of tetramer)-1 and a heat capacity change at the transition temperature of DeltaDNCp=14.9(+/-)3% kJ (mol of tetramerxK)-1. The two-state nature of the unfolding reaction is reflected in coinciding calorimetric and van't Hoff enthalpy values.

DSC studies of the conformational stability of barstar wild-type.

The temperature induced unfolding of barstar wild-type of bacillus amyloliquefaciens (90 residues) has been characterized by differential scanning microcalorimetry. The process has been found to be reversible in the pH range from 6.4 to 8.3 in the absence of oxygen. It has been clearly shown by a ratio of delta HvH/delta Hcal near 1 that denaturation follows a two-state mechanism. For comparison, the C82A mutant was also studied. This mutant exhibits similar reversibility, but has a slightly lower transition temperature. The transition enthalpy of barstar wt (303 kJ mol-1) exceeds that of the C82A mutant (276 kJ mol-1) by approximately 10%. The heat capacity changes show a similar difference, delta Cp being 5.3 +/- 1 kJ mol-1 K-1 for the wild-type and 3.6 +/- 1 kJ mol-1 K-1 for the C82A mutant. The extrapolated stability parameters at 25 degrees C are delta G0 = 23.5 +/- 2 kJ mol-1 for barstar wt and delta G0 = 25.5 +/- 2 kJ mol-1 for the C82A mutant.

Extreme thermostability of tarantula hemocyanin.

Biotops with extreme temperatures such as deserts force animals to avoid or escape high temperatures by biochemical, behavioural or morphological adaptation. In this context we tested the resistance to heat of the oxygen carrier hemocyanin from the ancient tarantula Eurypelma californicum, which is found in arid zones of North America. Differential scanning calorimetry, light scattering, crossed immunogelelectrophoresis and oxygen binding experiments show that the 24-meric hemocyanin is conformationally stable and fully functioning at temperatures up to 90 degrees C. Our results demonstrate that the cation-mediated state of oligomerization is not only crucial for the high cooperativity of oxygen binding of this hemocyanin, but also for its extreme stability in the physiological temperature and pH range.

Ca2+-independent interaction of annexin I with phospholipid monolayers.

At pH 6.0, the interaction of annexin I, a proteolytic fragment of annexin I and annexin V, was studied with monolayers composed of dipalmitoylphosphatidylserine (DPPS), dipalmitoylphosphatidylcholine (DPPC) or DPPS/DPPC mixtures (molar ratio 1:4). The measurements reveal that only annexin I shows a significant increase in the surface pressure at constant surface area in the absence of Ca2+ ions. We interpret these pressure changes as reflecting penetration of the protein. Kinetic analyses of the annexin I/monolayer interaction at pH 6.0 in the presence and absence of Ca2+ ions show differences between the interaction mechanisms that support the occurrence of a pH-regulated process. At pH 7.4, Ca2+ ions are required for the interaction.