Biomedical subjects

C Ebel

Publications and source records attributed to C Ebel.

At least 37 records · Page 2Linked to original sources

PubMed1999

Trans-splicing and cis-splicing in the colourless Euglenoid, Entosiphon sulcatum.

The colourless Euglenoid Entosiphon sulcatum is thought to have diverged before the symbiotic event which gave rise to the photosynthetic Euglenoid species as Euglena gracilis. We have isolated genes encoding spliced leader-sequence RNA (SL-RNA) and we show that pre-mRNAs are matured via a trans-splicing reaction in E. sulcatum, as in the case of E. gracilis. The 2.5-kb repeated DNA fragment which encodes the SL-RNA gene also encodes a 5S rRNA gene as well as the genes for the small nuclear (sn) RNAs U1, U2 and U5. The presence of snRNA U1 indicates that the classical cis-splicing mechanism also exists in E. sulcatum. In addition, we show that the E. sulcatum beta-tubulin gene has the intron borders GU-AG, typical of spliceosome-matured introns which have not yet been found in E. gracilis. The probable origins of trans- and cis-mechanisms in Euglenoids are discussed.

Animals↗

PubMed1999

Phosphorylation-induced dimerization of the FixJ receiver domain.

The 'two-component' transcriptional activator FixJ controls nitrogen fixation in Sinorhizobium meliloti. Phosphorylation of FixJ induces its dimerization, as evidenced by gel permeation chromatography and equilibrium sedimentation analysis. Phosphorylation-induced dimerization is an intrinsic property of the isolated receiver domain FixJN. Accordingly, chemical phosphorylation of both FixJ and FixJN are second-order reactions with respect to protein concentration. However, the second-order phosphorylation constant is 44-fold higher for FixJN than for FixJ. Therefore, the C-terminal transcriptional activator domain FixJC inhibits the chemical phosphorylation of the receiver domain FixJN. Conversely, FixJN has been shown previously to inhibit FixJC activity approximately 40-fold, reflecting the interaction between FixJN and FixJC. Therefore, we propose that modulation of FixJ activity involves both its dimerization and the disruption of the interface between FixJN and FixJC, resulting in the opening of the protein structure. Alanine scanning mutagenesis of FixJN indicated that the FixJ approximately P dimerization interface involves Val-91 and Lys-95 in helix alpha4. Dimerization was required for high-affinity binding to fixK promoter DNA.

Bacterial Proteins↗

PubMed1999

Oligomerization of the 17-kDa peptide-binding domain of the molecular chaperone HSC70.

Crystallographic and biochemical studies have indicated that the peptide-binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a beta-sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein. To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17-kDa protein (residues 385-540 of HSC70) consisting mainly of the peptide-binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60-kDa fragment of HSC70, which is composed of the 17-kDa domain and the 44-kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17-kDa peptide-binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide-binding site may be involved in the oligomerization process.

Adenosine Triphosphatases↗

PubMed1999

No risk of metal toxicity in combined spinal-epidural anesthesia.

UNLABELLED: Using the single level needle-through-needle technique for combined spinal-epidural anesthesia (CSE) may introduce very fine metal particles abraded by the spinal needle from the inner ground edge of the Tuohy needle into the patient. Either the local anesthetic administered epidurally or the peridural catheter may also pass intrathecally through the hole in the dura made by the spinal needle. To examine these concerns, the needle-through-needle technique was simulated in an in vitro model (18-gauge Tuohy needle; 27- or 29-gauge Quincke needle). The presence of abraded metal particles was identified by atomic absorption spectrography (AAS). The needles were then examined under an electron microscope. Metal particles could not be identified by using AAS in the needle-through-needle technique after normal clinical use, nor could traces of use be revealed by using an electron microscope to examine the Tuohy needle. With intentionally rough handling and caudal orientation of the spinal needle tip, minimal scratches could be seen by using an electron microscope, but there were no metal particles detected by AAS. In an anatomical preparation, the possible passage of the epidural catheter anesthetic through the dural puncture hole into the cerebrospinal fluid compartment was investigated endoscopically. Neither passage of dyed epidural local anesthetic nor penetration of the epidural catheter into the cerebrospinal fluid compartment could be demonstrated by endoscopy. We conclude that the needle-through-needle-technique is an acceptable way of performing CSE anesthesia. Endangering the patient by an unintentionally intrathecal misplacement of the epidural catheter seems to be very unlikely based on our in vitro model if small spinal needles (27- or 29-gauge) are used. IMPLICATIONS: Atomic absorption spectrography shows no contamination of the intrathecal compartment by abraded metal particles from the Tuohy needle by combined spinal-epidural anesthesia with the needle-through-needle technique. In vitro, neither passage of dyed epidural local anesthetic nor penetration of the epidural catheter into the cerebrospinal fluid compartment could be demonstrated by endoscopy.

Anesthesia, Epidural↗

PubMed1998

Evidence for kinetic intermediate states during the refolding of GdnHCl-denatured MM-creatine kinase. Characterization of a trapped monomeric species.

The kinetics of refolding of guanidinium chloride-denatured rabbit MM-creatine kinase was investigated. Recovery of enzymatic activity is biphasic, depending on the temperature but not on the protein or DTT concentration. Only 45% of the original, active dimeric form is recovered even after several hours of refolding. The reactivation yield is limited by the accumulation of a highly stable but nonproductive monomeric species. The ratio of "correct" to "incorrect" forms depends on the duration of exposure to the denaturant, which may be consistent with the existence of a heterogeneous population of unfolded states with regard to proline isomerization. The first fast reaction observed during renaturation results in the appearance of collapsed monomeric states, displaying features of a pre-molten globule state. These burst species are rapidly transformed into more structured monomers resembling a molten globule state possessing a partially folded C-terminal domain. A proportion of these latter transient intermediates (45%) associates into an active dimer, while the remainder (55%) is trapped by reshuffling in a monomeric dead-end product. Our results strongly indicate that (i) the dimeric state is a prerequisite for the expression of catalytic activity, (ii) the kinetic intermediates of refolding are very similar to those observed during equilibrium unfolding, and (iii) refolding of creatine kinase in these conditions is limited by the accumulation of inactive misfolded nondimerizable monomer.

Anilino Naphthalenesulfonates↗

PubMed1998

Solution structure of the ternary complex between aminoacyl-tRNA, elongation factor Tu, and guanosine triphosphate.

Complex formation between elongation factor Tu (EF-Tu), Phe-tRNAPhe, and GTP was analyzed by small-angle neutron and X-ray scattering methods. Both techniques show that the ternary complex consists of one EF-Tu and one aminoacyl-tRNA. No shift in stoichiometry was detected when the temperature was raised from 5 to 37 degreesC, in contrast to previous observations obtained from RNase A protection experiments [Bilgin and Ehrenberg (1995) Biochemistry34, 715-719]. A small but significant increase in the radius of gyration of the complex was observed when the temperature was decreased from 37 to 5 degreesC. The X-ray solution scattering patterns were compared with those calculated from the crystal structure of the complex formed between EF-Tu from Thermus aquaticus and Phe-tRNAPhe from yeast. The comparison shows that the solution structure of the ternary complex, formed entirely from Escherichia coli components and under translationally optimal buffer conditions, is very close to the crystal structure, formed from heterologous components under very different conditions. Furthermore, for the hybrid complex in solution there is no evidence for the formation of trimers as suggested by the crystal structure.

Binding Sites↗

PubMed1998

Oligomeric structure of the repressor of the bacteriophage Mu early operon.

The regulation of the lytic and lysogenic development in the life cycle of bacteriophage Mu is regulated in part by its repressor, c, which binds to three operator sites, O1, O2 and O3, overlapping two divergent promoters. The oligomeric structure of this repressor protein was investigated by hydrodynamic and biochemical methods. Size-exclusion chromatography, analytical ultracentrifugation, dynamic light scattering, crosslinking and direct electron microscopy observations suggest that c exists primarily as a hexamer with a molecular mass of 120-140 kDa at low concentrations, i.e. in the 10-microM range. This molecule undergoes a self-assembly process leading to dodecamers and higher order species as the concentration is further increased in a manner depending on the nature of the solvent. Our results also suggest that these species have an elongated structure, and a possible arrangement of the subunits within the hexamer is proposed. The implication of this unusual quaternary structure for a repressor in its interaction with the operator sites O1 and O2 remains to be elucidated.

Bacteriophage mu↗

PubMed1998

In vitro investigation of cerebrospinal fluid leakage after dural puncture with various spinal needles.

UNLABELLED: Postspinal headache is one of the most common complications of spinal anesthesia and has repeatedly led to controversy concerning needle size and configuration. In an in vitro investigation, we measured cerebrospinal fluid (CSF) leakage with Sprotte, Whitacre, Quincke, and Atraucan needles under physiological conditions in human dura. The puncture characteristics were examined under an electron microscope. The pencil-point needles show 2-3 times less leakage of CSF compared with the cutting Quincke needles of corresponding size. Between the Sprotte and the Whitacre needles, there were no significant differences. The least loss of CSF occurred with the 26-gauge Atraucan needle. Under the electron microscope, a sharply delineated, persistent perforation channel was shown with the Quincke needles, which may explain the high CSF loss. With pencil-point needles, which push the tissue apart bluntly, a large opening on the inside is found, with some tearing of the dura. However, in contrast to the cutting needles, a persistent perforation channel is not manifested. The 26-gauge Atraucan needle, which both cuts and pushes apart conically, shows a relatively discrete opening on the inside, with slight tears in the dura and arachnoidea but without a visible perforation channel. The results of our study show that larger needles (26-gauge Atraucan) that are easier to handle can lead to good and, in some cases even better, puncture results if they have characteristics of both the cutting and the pencil-point needles. IMPLICATIONS: We compared several brands of pencil-point and standard cutting spinal needles of varying sizes. All pencil-point needles had less cerebrospinal fluid leakage, the least loss occurring with 26-gauge Atraucan needles. Electron microscopic examination of the dura after puncture showed characteristic findings with each needle type. We conclude that the combined cutting and pencil-point characteristics seen in the Atraucan needle may have clinical advantages.

Cerebrospinal Fluid↗

PubMed1998

Intrathoracic and pulmonary blood volume during CO2-pneumoperitoneum in humans.

BACKGROUND: Induction of CO2-pneumoperitoneum may have significant effects on systemic and pulmonary haemodynamics. We hypothesized that intrathoracic (ITBV) and pulmonary blood volume (PBV) are affected during intra-abdominal CO2-insufflation, which may be pronounced by positional changes of the patient. METHODS: Sixteen anaesthetized patients were studied before, during and after CO2-pneumoperitoneum for laparoscopic cholecystectomy. A dye indicator technique was used to assess ITBV and PBV. In addition, gas exchange and haemodynamics were recorded. RESULTS: In the supine position, induction of CO2-pneumoperitoneum had no effects on ITBV, PBV and cardiac output. Mean systemic arterial pressure increased from 10.9 +/- 1.5 kPa (82 +/- 11 mmHg) to 12.7 +/- 1.5 kPa (95 +/- 11 mmHg, P < 0.01). In the reverse Trendelenburg position ITBV decreased from 19.8 +/- 5.1 ml.kg-1 to 16.7 +/- 3.7 ml.kg-1 (P < 0.05) during CO2-insufflation, but increased to control values after 20 min. PBV decreased from 4.2 +/- 1.2 ml.kg-1 to 3.4 +/- 1.1 ml.kg (P < 0.05) and remained decreased during CO2-pneumoperitoneum. Calculated venous admixture was unchanged throughout the study. Deflation of CO2-pneumoperitoneum increased ITBV (22.4 +/- 5.2 ml.kg-1, P < 0.05) and cardiac output above control values. CONCLUSIONS: In anaesthetized-paralyzed patients in the reverse Trendelenburg position intra-abdominal CO2-insufflation is associated with significant alterations of ITBV and PBV. The release of CO2-pneumoperitoneum is associated with a re-distribution of blood into the thorax.

Abdomen↗

PubMed1997

A central role for water in the control of the spin state of cytochrome P-450scc.

A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.

Centrifugation↗

PubMed1997

Effect of thoracic epidural anaesthesia on ventilation-perfusion distribution and intrathoracic blood volume before and after induction of general anaesthesia.

BACKGROUND: Gas exchange is impaired during general anaesthesia due to development of shunt and ventilation-perfusion mismatching. Thoracic epidural anaesthesia (TEA) may affect the mechanics of the respiratory system, intrathoracic blood volume and possibly ventilation-perfusion (VA/Q) distribution during general anaesthesia. METHODS: VA/Q relationships were analyzed in 24 patients undergoing major abdominal surgery. Intrapulmonary shunt (Qs/QT), perfusion of "low" VA/Q areas, ventilation of "high" VA/Q regions, dead space ventilation and mean distribution of ventilation and perfusion were calculated from the retention/excretion data of six inert gases. Intrathoracic blood volume (ITBV) and pulmonary blood volume (PBV) were determined with a double indicator technique. Recordings were made before and after administration of 8.5 +/- 1.5 ml bupivacaine 0.5% (n = 12) or 8.3 +/- 1.8 ml placebo (n = 12) into a thoracic epidural catheter and after induction of general anaesthesia. RESULTS: Before TEA, Qs/QT was normal in the bupivacaine group (2 +/- 2%) and the placebo group (2 +/- 3%). TEA covering the dermatomal segments T 12 to T 4 had no effect on VA/Q relationships, ITBV and PBV. After induction of general anaesthesia Qs/QT increased to 8 +/- 4% (bupivacaine group, P < 0.05 and to 7 +/- 2% (placebo group, P < 0.05). ITBV and PBV decreased significantly to the same extent in the bupivacaine group and the placebo group. CONCLUSIONS: TEA has no effect on VA/Q distribution, gas exchange and intrathoracic blood volume in the awake state and does not influence development of Qs/QT and VA/Q inequality after induction of general anaesthesia.

Anesthesia, Epidural↗

PubMed1996

An example of non-conservation of oligomeric structure in prokaryotic aminoacyl-tRNA synthetases. Biochemical and structural properties of glycyl-tRNA synthetase from Thermus thermophilus.

Glycyl-tRNA synthetase (Gly-tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five-step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D.T., Mazauric, M.-H., Kern, D. & Moras, D. (1995) EMBO J. 14, 4156-4167]. Molecular-mass determinations of the native and denatured protein indicate an oligomeric structure of the alpha 2 type consistent with that found for eukaryotic Gly-tRNA synthetases (yeast and Bombyx mori), but different from that of Gly-tRNA synthetases from mesophilic prokaryotes (Escherichia coli and Bacillus brevis) which are alpha 2 beta 2 tetramers. N-terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes (B. mori, Homo sapiens, yeast and Caenorhabditis elegans) but no significant identity was found with both alpha and beta chains of the prokaryotic enzymes (E. coli, Haemophilus influenzae and Coxiella burnetii) albeit the enzyme is deprived of the N-terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly-tRNA synthetase combines strong structural homologies of eukaryotic Gly-tRNA synthetases with a feature of prokaryotic synthetases. Heat-stability measurements show that this synthetase keeps its ATP-PPi exchange and aminoacylation activities up to 70 degrees C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA(Gly) does not induce protection. Cross-aminoacylations reveal that the thermophilic Gly-tRNA synthetase charges heterologous E. coli tRNA(gly(GCC)) and tRNA(Gly(GCC)) and yeast tRNA(Gly(GCC)) as efficiently as T. thermophilus tRNA(Gly). All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly-tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three-dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.

Amino Acid Sequence↗

PubMed1996

The expression of E.coli threonyl-tRNA synthetase is regulated at the translational level by symmetrical operator-repressor interactions.

Threonyl-tRNA synthetase from Escherichia coli represses the translation of its own mRNA by binding to the operator region located upstream from the ribosome binding site. The operator contains two stemloop structures which interact specifically with the homodimeric enzyme. Here, we provide in vitro and in vivo evidence that these two stem-loop structures are recognized by the enzyme in an analogous way and mimic the anticodon arm of E.coli tRNA(Thr). Determination of the stoichiometry of the different RNA-threonyl-tRNA synthetase complexes reveals that two tRNA(Thr) molecules bind to the enzyme whereas only one thrS operator interacts with the homodimeric enzyme. A model is presented in which the two anticodon-like domains of the operator bind symmetrically to the two tRNA(Thr) anticodon recognition sites (one per subunit) of the dimeric threonyl-tRNA synthetase. Although symmetrical operator-repressor interactions in transcriptional control are widespread, this report stresses the importance of such interactions in translational regulation of gene expression.

Anticodon↗

PubMed1996

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Allosteric Regulation↗

PubMed1995

Reversible dissociation and unfolding of dimeric creatine kinase isoenzyme MM in guanidine hydrochloride and urea.

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far-ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1-anilinonaphthalene-8-sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non-coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far-ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near-ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4M). These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.

Anilino Naphthalenesulfonates↗

PubMed1995

Aggregation of VSV M protein is reversible and mediated by nucleation sites: implications for viral assembly.

Purified M protein of VSV has been reported to aggregate at low NaCl concentration. Using light scattering, analytical centrifugation, and electron microscopy (EM), we have studied this phenomenon. Our results demonstrate that self aggregation of M protein can be reversed by increasing the salt concentration. Below 250 mM NaCl, there is an equilibrium between aggregates and monomeric M protein. Most importantly, we demonstrate that aggregation only occurs in the presence of nucleation sites and that these sites are sensitive to trypsin. We have found conditions under which these nucleation sites can be eliminated, after which M remains soluble even at low salt concentration. Finally, using EM, we show that the aggregates of purified M protein share common structural aspects with the previously described internal "cigar" around which the nucleocapsid is wrapped. These new results help to explain why M is a soluble protein in the cytoplasm of the infected cell just up to the moment that it is integrated into the budding virion.

Animals↗

PubMed1995

Solution structure of glyceraldehyde-3-phosphate dehydrogenase from Haloarcula vallismortis.

The subunit molecular mass of glyceraldehyde-3-phosphate dehydrogenase from the extreme halophile Haloarcula vallismortis (hGAPDH) was determined by mass spectrometry to be 35990 +/- 80 daltons, similar to other GAPDHs. Complementary density, sedimentation and light scattering experiments showed the protein to be a tetramer that binds 0.18 +/- 0.10 gram of water and 0.07 +/- 0.02 gram of KCl per gram of protein, in multimolar KCl solutions. At low salt (below 1 M), the tetramer dissociated into unfolded monomers. This is the third halophilic protein for which solvent interactions were measured. The extent of these interactions depends on the protein, but all form an invariant particle, in multimolar NaCl or KCl solutions, that binds a high proportion of salt when compared to non-halophilic proteins.

Journal Article↗

PubMed1994

Cold-induced conformational changes of ribonuclease A as investigated by subzero transverse temperature gradient gel electrophoresis.

Subzero temperature gradient gel electrophoresis is a new approach which allows to measure the transition temperature of low temperature-induced subtle conformational changes of proteins and to detect the different conformational states, including unfolded states. Using this technique under destabilizing conditions, i.e., in the presence of 4 M urea, bovine pancreas ribonuclease A exhibited two transitions: (i) a continuous transition with a midpoint temperature of -14 degrees C corresponding to a rapid equilibrium between the initial enzyme state and a conformational state more compact than the initial one; (ii) a discontinuous transition at -22.5 degrees C from intermediate to a non migrating species. Under reducing conditions this second transition was shifted toward high temperatures (-18.5 degrees C). We attempted to detect these two transitions by differential scanning calorimetry, UV spectrophotometry and circular dichroism measurements. These transitions have been ascribed to subtle cold-induced conformational changes.

Calorimetry, Differential Scanning↗