Transcatheter recanalisation and stenting of a closed ductus arteriosus in duct dependent lung perfusion.
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Ductus Arteriosus, Patent↗
Biomedical subjects
F X Schmid
Publications and source records attributed to F X Schmid.
Prolyl isomerases do not catalyze isomerization of non-prolyl peptide bonds.
Prolyl isomerases accelerate the cis <--> trans isomerization of prolyl peptide bonds during protein folding and probably also in folded proteins. We asked whether this catalytic function is in fact restricted to prolyl bonds or whether the isomerizations of 'normal' non-prolyl peptide bonds are catalyzed as well. By using the P39A variant of ribonuclease T1 as a substrate we find that the trans --> cis isomerization of the Tyr38-Ala39 bond in the refolding of this protein is not catalyzed by prolyl isomerases of the cyclophilin, FKBP and parvulin families. These enzymes are neither able to catalyze amide bond isomerizations in the proline-free model peptide Ala-Ala-Tyr-Ala-Ala.
Amides↗
Clinical performance of the native pulmonary valve in the systemic circulation.
BACKGROUND AND AIM OF THE STUDY: In a number of corrective and palliative procedures the autologous pulmonary valve is used as the systemic semilunar valve. This study reviews the surgical results and function of the native pulmonary valve in the systemic position after various surgical procedures. METHODS: Between January 1994 and December 1997, the autologous pulmonary valve was transferred functionally or anatomically into the systemic circulation in 89 patients. Follow up echocardiograms and cardiac angiograms were reviewed for 51 neonates with transposition of the great arteries after an arterial switch operation (ASO), in 21 patients after first-stage palliation of hypoplastic left heart syndrome (HLHS), in eight children and adults with pulmonary autograft aortic valve replacement (Ross procedure), and in nine patients with a pulmonary artery-to-aortic anastomosis (Damus-Kaye-Stansel (DKS) procedure) in complex heart defects with outflow obstruction. RESULTS: Nine patients (five with HLHS) died; thus, overall mortality rate was 10.2%. There was no evidence of valve-related mortality. Trivial insufficiency following ASO was noted in 11 patients, with no progression of incompetence over time. None of the HLHS patients had pulmonary insufficiency preoperatively, but all showed mild regurgitation on postoperative echocardiography. There was a moderate increase in insufficiency which was attenuated after an early second-stage palliation. Three of nine patients undergoing a DKS anastomosis demonstrated a hemodynamically insignificant insufficiency. Modification of the surgical technique avoided postoperative regurgitation. Four of seven patients having a Ross procedure showed trivial but non-progressive neoaortic regurgitation. CONCLUSIONS: Based on this experience, the autologous pulmonary valve performs adequately at intermediate term follow up. Postoperatively, trivial regurgitation was a frequent finding but was hemodynamically insignificant. Progression or late development of insufficiency as well as stenosis were rare problems.
Adolescent↗
Catalysis of protein folding by parvulin.
Recently a new family of prolyl isomerases was discovered, which is unrelated with the cyclophilins or the FK-506 binding proteins. Parvulin, the smallest member of this new family, is a protein with only 92 residues, but parvulin-like domains occur in several large proteins that are apparently involved in protein folding or activation processes. We show here that, in addition to its activity in assays with proline-containing tetrapeptides, parvulin catalyzes the proline-limited folding of a variant of ribonuclease T1 with a kcat/Km value of 30,000 M-1 s-1. This value is much smaller than the kcat/Km value of 1.1x10(7) M-1 s-1 determined for the parvulin-catalyzed prolyl isomerization in the tetrapeptide succinyl-Ala-Leu-Pro-Phe-4-nitroanilide. Parvulin itself unfolds and refolds reversibly in a simple two-state reaction with a Gibbs free energy of stabilization of 28 kJ/mol at 10 degrees C. Most of the unfolded parvulin molecules refold in a slow reaction that involves prolyl isomerization and is catalyzed by cyclophilin, another prolyl isomerase. Moreover, parvulin accelerates its own refolding in an autocatalytic fashion, and the rate of refolding increases tenfold when the concentration of parvulin is increased from 0.5 to 3.0 microM. Apparently, small single-domain prolyl isomerases catalyze prolyl isomerization much better in short peptides than in protein folding reactions, presumably because the prolyl bonds are less accessible in refolding protein chains. It is possible that the additional domains of the large prolyl isomerases improve the affinity for protein substrates.
Catalysis↗
Thermodynamics of the complex protein unfolding reaction of barstar.
The complex unfolding reaction of barstar has been characterized by studying the apparent rate of unfolding, monitored by intrinsic Trp fluorescence, as a function of temperature and guanidine hydrochloride (GdnHCl) concentration. The kinetics of unfolding and folding of wild-type (wt) barstar at 5 degrees C were first studied in detail. It is shown that when unfolding is carried out using concentrations of GdnHCl in the posttransition zone of unfolding, the change in fluorescence that accompanies unfolding occurs in two phases: 30% of the change occurs in a burst phase that is complete within 4 ms, and 70% of the change occurs in a fast phase that is complete within 2 s. In contrast, when the protein is unfolded at 25 degrees C, no burst-phase change in fluorescence is observed. To confirm that a burst-phase change in fluorescence indeed accompanies unfolding at low temperature, unfolding studies were also carried out on a marginally destabilized mutant form of barstar for which the burst-phase change in fluorescence is shown to be as high as 70%. These results confirm a previous report [Nath et al., (1996), Nat. Struct. Biol. 3, 920-923], in which the detection of a burst-phase change in circular dichroism at 222 nm during unfolding at 25 degrees C led to the inclusion of a rapidly formed kinetic intermediate, IU, on the unfolding pathway. To characterize thermodynamically the unfolding pathway, apparent unfolding rates were then measured at six different concentrations of GdnHCl in the range 2.6 to 5.0 M, at five different temperatures from 5 to 46 degrees C. The subsequent analysis was done on the basis of the observation that a preequilibrium between the fully folded state (F) and IU gets established rapidly before further unfolding to the completely unfolded state (U). The results indicate that IU has a specific heat capacity similar to that of F and therefore suggest that IU is as compact as F, with practically no exposure of the hydrophobic core. On the other hand, the transition state of unfolding has a 45% greater heat capacity than F, indicating that significant hydration of the hydrophobic core occurs only after the rate-limiting step of unfolding.
Bacillus↗
Modular structure of the trigger factor required for high activity in protein folding.
The Escherichia coli trigger factor is a peptidyl-prolyl cis/trans isomerase (PPIase) which catalyzes proline-limited protein folding extremely well. It has been found associated with nascent protein chains as well as with the chaperone GroEL. The trigger factor utilizes protein regions outside the central catalytic domain for catalyzing refolding of unfolded proteins efficiently. Here we produced several fragments which encompass individual domains or combinations of the middle FKBP-like domain (M) with the N-terminal (N) and C-terminal (C) regions, respectively. These fragments appear to be stably folded. They show ordered structure and cooperative urea-induced unfolding transitions, and the far-UV CD spectrum of the intact trigger factor is well represented by the sum of the spectra of the fragments. This suggests that the native trigger factor shows a modular structure, which is composed of three fairly independent folding units. In the intact protein there is a slight mutual stabilization of these units. The high enzymatic activity in protein folding could not be restored by fusing alternatively the N or the C-terminal regions to the catalytic domain (in NM and MC constructs, respectively). Surprisingly, the high folding activity of the intact trigger factor has been regained partially by functional complementation of the overlapping NM and MC constructs.
Amino Acid Isomerases↗
Cyclophilin active site mutants have native prolyl isomerase activity with a protein substrate.
The prolyl isomerase activity of cyclophilins is traditionally measured by an assay in which prolyl cis/trans isomerization in a chromogenic tetrapeptide is coupled with its isomer-specific cleavage by chymotrypsin. Two variants of mitochondrial cyclophilin with substitutions in the presumed active site (R73A and H144Q) are inactive in the protease-coupled assay, but show almost wild-type activity in an assay that is based on the catalysis of a proline-limited protein folding reaction. This prolyl isomerase assay is preferable, both because coupling with proteolysis is avoided and because an intact protein instead of a short peptide is used as a substrate. Possibly, some earlier conclusions about the catalytic mechanism and the involvement of the prolyl isomerase activity in the cellular function of immunophilins may need reevaluation.
Amino Acid Isomerases↗
The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis.
Class I heat-inducible genes in Bacillus subtilis consist of the heptacistronic dnaK and the bicistronic groE operon and form the CIRCE regulon. Both operons are negatively regulated at the level of transcription by the HrcA repressor interacting with its operator, the CIRCE element. Here, we demonstrate that the DnaK chaperone machine is not involved in the regulation of HrcA and that the GroE chaperonin exerts a negative effect in the post-transcriptional control of HrcA. When expression of the groE operon was turned off, the dnaK operon was significantly activated and large amounts of apparently inactive HrcA repressor were produced. Overproduction of GroEL, on the other hand, resulted in decreased expression of the dnaK operon. Introduction of the hrcA gene and its operator into Escherichia coli was sufficient to elicit a transient heat shock response, indicating that no additional Bacillus-specific gene(s) was needed. As in B. subtilis, the groEL gene of E. coli negatively influenced the activity of HrcA. HrcA could be overproduced in E. coli, but formed inclusion bodies which could be dissolved in 8 M urea. Upon removal of urea, HrcA had a strong tendency to aggregate, but aggregation could be suppressed significantly by the addition of GroEL. Purified HrcA repressor was able specifically to retard a DNA fragment containing the CIRCE element, and the amount of retarded DNA was increased significantly in the presence of GroEL. These results suggest that the GroE chaperonin machine modulates the activity of the HrcA repressor and therefore point to a novel function of GroE as a modulator of the heat shock response.
Bacillus subtilis↗
Diffusion control in an elementary protein folding reaction.
The cold-shock protein CspB (from Bacillus subtilis), a very small protein of 67 residues, folds extremely fast in a reversible N &lrharr; U two-state reaction. Both unfolding and refolding are strongly decelerated when the viscosity of the solvent is increased by adding ethylene glycol or sucrose. The folding of CspB thus seems to follow Kramers' model for reactions in which the reactants must diffuse together. It indicates that the compaction of the protein chain occurs in the rate-limiting step of folding. Chain diffusion to a productively collapsed form and the crossing of a high energy barrier are thus tightly coupled in this folding reaction, and the measured reaction rate depends on both the diffusion of the protein chain in the solvent and the magnitude of the activation energy. We suggest that in protein folding an energetic barrier is essential to separate the native from the unfolded conformations of a protein. This barrier protects the ordered structure of a native protein against continuous unfolding by diffusive chain motions and leads to apparent two-state behavior.
Bacillus subtilis↗
Combinations of the alpha-helix-turn-alpha-helix motif of TetR with respective residues from LacI or 434Cro: DNA recognition, inducer binding, and urea-dependent denaturation.
We constructed 10 different variants of TetR by substituting all or some of the residues in the alpha-helix-turn-alpha-helix (HTH) operator binding motif with the respective amino acids from LacI or 434Cro. The variants were soluble, negative transdominant over tetR in vivo, and as active as wild-type TetR in tetracycline binding in vitro. The urea-induced denaturation of the 10 variants occurs in single reversible transitions, which are centered around 4.3 M urea. Denaturation is concentration-dependent, supporting a simple two-state mechanism in which the folded dimeric protein is in equilibrium with unfolded monomers. An analysis according to the two-state model yields a Gibbs free energy of stabilization (at 0 M urea, 25 degrees C) of about 75 kJ/mol, typical for dimeric proteins of this size. Even a deletion of 24 residues from the reading head decreased the stability by only 2.7 kJ/mol. These results suggest that the DNA reading head of Tet repressor is a thermodynamically independent domain and that the thermodynamic stability of the Tet repressor dimer is determined by the association of the dimerization domains of the individual monomers. Variants containing replacements in the first alpha-helix of HTH did not show any DNA binding activity whatsoever. We attribute this to the alteration of the two N-terminal residues in this alpha-helix. TetR variants were active in nonspecific DNA binding, when either all or only the solvent-exposed residues in the recognition alpha-helix of HTH were exchanged to the respective LacI sequence. Replacement of the same residues by the respective amino acids from 434Cro yielded hybrid proteins that specifically recognize tetO in vitro. Taken together, these results establish that the similarity of operator recognition between 434Cro and TetR is greater than between TetR and LacI and confirm that prediction of the recognized DNA sequence is not obvious from the sequence of the respective HTH or recognition alpha-helix.
Amino Acid Sequence↗
Cooperation of enzymatic and chaperone functions of trigger factor in the catalysis of protein folding.
The trigger factor of Escherichia coli is a prolyl isomerase and accelerates proline-limited steps in protein folding with a very high efficiency. It associates with nascent polypeptide chains at the ribosome and is thought to catalyse the folding of newly synthesized proteins. In its enzymatic mechanism the trigger factor follows the Michaelis-Menten equation. The unusually high folding activity of the trigger factor originates from its tight binding to the folding protein substrate, as reflected in the low Km value of 0.7 microM. In contrast, the catalytic constant kcat is small and shows a value of 1.3 s(-1) at 15 degrees C. An unfolded protein inhibits the trigger factor in a competitive fashion. The isolated catalytic domain of the trigger factor retains the full prolyl isomerase activity towards short peptides, but in a protein folding reaction its activity is 800-fold reduced and no longer inhibited by an unfolded protein. Unlike the prolyl isomerase site, the polypeptide binding site obviously extends beyond the FKBP domain. Together, this suggests that the good substrate binding, i.e. the chaperone property, of the intact trigger factor is responsible for its high efficiency as a catalyst of proline-limited protein folding.
Amino Acid Isomerases↗
Evaluation of gastric intramucosal pH during and after pediatric cardiac surgery.
OBJECTIVES: In adult patients, intramucosal pH (pHi) has been advocated to detect postoperative complications. The purpose of our study was to evaluate this technique in pediatric patients during and after cardiac surgery. METHODS: Thirty-five infants (age: 5 days to 15 years, median 1.8 years; and weight: 3.2-32 kg, median 9.8 kg) were studied. pHi was measured before cardiopulmonary bypass (CPB), after 30 min of CPB, prior to weaning off CPB, at intensive care unit arrival, and 6, 12, 24, 48 and 72 h after surgery. RESULTS: There were no complications related to the tonometer. A pathologically low pHi < 7.32 was found during surgery in less than 17%, at intensive care unit arrival in 83% and after 48 h in 18%. pHi values were lower (P < 0.05) at intensive care unit arrival (7.25 +/- 0.08) and after 6 h (7.28 +/- 0.09) than afterwards. pHi correlated with arterial pH (r = 0.66), central-peripheral temperature difference (r = -0.36), lactate (r = -0.32) and central venous pressure (r = -0.21). Patients after a Fontan procedure had postoperatively a lower pHi than after other operations (P < 0.05). None of the patients died or developed organ failure. Six patients had signs of organ dysfunction. Their pHi (median 7.23, range 7.14-7.28) could not differentiate them from the other patients. CONCLUSIONS: With current equipment, tonometry cannot be recommended for the management of pediatric patients after cardiac surgery. However, as a semi-invasive method tonometry deserves further evaluation.
Adolescent↗
Functions of FKBP12 and mitochondrial cyclophilin active site residues in vitro and in vivo in Saccharomyces cerevisiae.
Cyclophilin and FK506 binding protein (FKBP) accelerate cis-trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5-20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 "active-site" mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.
Binding Sites↗
Thermodynamic properties of an extremely rapid protein folding reaction.
The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to avoid misfolding prior to the rate-limiting step, and a native-like activated state reduces the risk of non-productive side reactions during the final steps to the native state.
Bacillus subtilis↗
The role of a trans-proline in the folding mechanism of ribonuclease T1.
Protein folding is often retarded by the cis reversible trans isomerizations of prolyl peptide bonds both in vitro and in vivo. An important role for the folding mechanism is well established for the prolyl peptide bonds that are cis in the native protein, but not for those that are trans. Here we investigated the role of trans-Pro73 for the folding of ribonuclease T1 (which additionally contains two cis-prolines) by comparing the wild-type protein with the Pro73-->Val variant. The Pro-->Val substitution led to a destabilization of the folded protein by 8.5 kJ/mol, which is explained by the strong, 25-fold increase in the rate of unfolding. In contrast, the rates and amplitudes of the fast and slow refolding reactions were virtually unchanged. trans-Proline residues remain largely trans after unfolding, and therefore their contributions to the observed folding kinetics should indeed be insignificant for proteins which also contain one or more cis prolines. The cis-proline residues dominate the kinetics of refolding, because almost all slow-folding molecules contain the respective incorrect (trans) isomers, and because trans-->cis isomerizations are slower than cis-->trans isomerizations. The inability to detect contributions from a trans-proline to the kinetics of folding does not imply that this proline is non-essential for folding in the sense that its cis reversible trans isomerization is energetically uncoupled from conformational folding.
Enzyme Stability↗
Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase.
Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.
Disulfides↗
A thermodynamic coupling mechanism for GroEL-mediated unfolding.
Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high.
Adenosine Diphosphate↗
Autocatalytic folding of the folding catalyst FKBP12.
Prolyl isomerases are folding enzymes and thus have the potential to catalyze their own folding. We show here that the folding of cytosolic FKBP12 (FK 506 binding protein) is an autocatalytic process both for the mature protein and for a fusion protein with an amino-terminal extension of 16 residues. Native FKBP contains seven trans-prolyl peptide bonds, and the cis-to-trans isomerizations of some or all of them constitute the slow, rate-limiting events in folding. The rate of an autocatalytic reaction increases with reactant concentration, because the product catalyzes its own formation. Accordingly, the folding of the fusion protein was more than 10-fold accelerated when the protein concentration was increased from 0.05 microM to 10 microM. At high concentrations of both forms of FKBP12 autocatalysis was very efficient, and the observed folding rate seemed to approach the rate of the fast direct folding reaction of the protein molecules with the correct (all trans) peptidyl-prolyl bond conformation.
Amino Acid Isomerases↗