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F X Schmid

Publications and source records attributed to F X Schmid.

At least 109 records · Page 6Linked to original sources
PubMed1992

Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1.

The folding and unfolding kinetics within the transition region were measured for RNase A and for RNase T1. The data were used to evaluate the theoretical models for the influence of prolyl isomerization on the observed folding kinetics. These two proteins were selected, since the folding reaction of RNase A is faster than prolyl isomerization, whereas in RNase T1, folding is slower than isomerization in the transition region. Folding of RNase T1 was investigated for three variants with different numbers of cis prolyl residues. The results indicate that in the transition region the folding rates are indeed strongly dependent on the number of prolyl residues. The variant of RNase T1 that contains only one cis prolyl residue folds about ten times faster than two variants that contain two cis prolyl residues. For both RNase A and RNase T1, the apparent rates of folding and unfolding as well as the corresponding amplitudes depend on the concentration of denaturant in a manner that was predicted by the model calculations. When refolding was started from the fast-folding species, additional kinetic phases could be observed in the transition region for both proteins. The obtained values could be used to calculate the microscopic rate constants of folding and isomerization on the basis of theoretical models.

Exoribonucleases↗
PubMed1992

Stability, quaternary structure, and folding of internal, external, and core-glycosylated invertase from yeast.

The role of carbohydrate chains for the structure, function, stability, and folding of glycoproteins has been investigated using invertase as a model. The protein is encoded by several different genes, and its carbohydrate moiety is heterogeneous. Both properties complicate physicochemical comparisons. Here we used the temperature-sensitive sec18 secretion mutant of yeast with a single invertase gene (SUC2). This mutant produces the carbohydrate-free internal invertase, the core-glycosylated form, and, at the permissive temperature, the fully glycosylated external enzyme, all with identical protein moieties. The core-glycosylated enzyme resembles the nascent glycoprotein chain that folds in the endoplasmic reticulum. Therefore, it may be considered a model for the in vivo folding of glycoproteins. In addition, because of its uniform glycosylation, it can be used to investigate the state of association of native invertase. Glycosylation is found to stabilize the protein with respect to thermal denaturation and chaotropic solvent components; the stabilizing effect does not differ for the external and the core-glycosylated forms. Unlike the internal enzyme, the glycosylated forms are protected from aggregation. Native internal invertase is a dimer (115 kDa) whereas the core-glycosylated enzyme is a mixture of dimers, tetramers, and octamers. This implies that core-glycosylation is necessary for oligomerization to tetramers and octamers. Dimerization is required and sufficient to generate enzymatic activity; further association does not alter the specific activity of core-glycosylated invertase, suggesting that the active sites of invertase are not affected by the association of the dimeric units. Reconstitution of the glycosylated and nonglycosylated forms of the enzyme after preceding guanidine denaturation depends on protein concentration. The maximum yield (approximately 80%) is obtained at pH 6-8 and protein concentrations < or = 4 micrograms/mL for the nonglycosylated and < or = 40 for the glycosylated forms of the enzyme. The lower stability of the internal enzyme is reflected by a narrower pH range of reactivation and enhanced aggregation. As indicated by the sigmoidal reactivation kinetics at low protein concentration both folding and association are rate-determining.

Enzyme Reactivators↗
PubMed1992

Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation.

Ribonuclease A is known to form an equilibrium mixture of fast-folding (UF) and slow-folding (US) species. Rapid unfolding to UF is then followed by a reaction in the unfolded state, which produces a mixture of UF, USII, USI, and possibly also minor populations of other US species. The two cis proline residues, P93 and P114, are logical candidates for producing the major US species after unfolding, by slow cis <==> trans isomerization. Much work has been done in the past on testing this proposal, but the results have been controversial. Site-directed mutagenesis is used here. Four single mutants, P93A, P93S, P114A, and P114G, and also the double mutant P93A, P114G have been made and tested for the formation of US species after unfolding. The single mutants P114G and P114A still show slow isomerization reactions after unfolding that produce US species; thus, Pro 114 is not required for the formation of at least one of the major US species of ribonuclease A. Both the refolding kinetics and the isomerization kinetics after unfolding of the Pro 93 single mutants are unexpectedly complex, possibly because the substituted amino acid forms a cis peptide bond, which should undergo cis --> trans isomerization after unfolding. The kinetics of peptide bond isomerization are not understood at present and the Pro 93 single mutants cannot be used yet to investigate the role of Pro 93 in forming the US species of ribonuclease A. The double mutant P93A, P114G shows single exponential kinetics measured by CD, and it shows no evidence of isomerization after unfolding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

Amino Acid Sequence↗
PubMed1992

Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate.

The replacement of tryptophan 59 of ribonuclease T1 by a tyrosine residue does not change the stability of the protein. However, it leads to a strong acceleration of a major, proline-limited reaction that is unusually slow in the refolding of the wild-type protein. The distribution of fast- and slow-folding species and the kinetic mechanism of slow folding are not changed by the mutation. Trp-59 is in close contact to Pro-39 in native RNase T1 and probably also in an intermediate that forms rapidly during folding. We suggest that this specific interaction interferes with the trans----cis reisomerization of the Tyr-38-Pro-39 bond at the stage of a native-like folding intermediate. The steric hindrance is abolished either by changing Trp-59 to a less bulky residue, such as tyrosine, or, by a destabilization of folding intermediates at increased concentrations of denaturant. Under such conditions folding of the wild-type protein and of the W59Y variant no longer differ. These results provide strong support for the proposal that trans----cis isomerization of Pro-39 is responsible for the major, very slow refolding reaction of RNase T1. They also indicate that specific tertiary interactions in folding intermediates do exist, but do not necessarily facilitate folding. They can have adverse effects and decelerate rate-limiting steps by trapping partially folded structures.

Models, Molecular↗
PubMed1991

Catalysis of protein folding by cyclophilins from different species.

Cyclophilins are a class of ubiquitous proteins with yet unknown function. They were originally discovered as the major binding proteins for the immunosuppressant cyclosporin A. The only known catalytic function of these proteins in vitro is the cis/trans isomerization of Xaa-Pro bonds in oligopeptides. This became clear after the discovery that bovine cyclophilin is identical with porcine prolyl isomerase. This enzyme accelerates slow, proline-limited steps in the refolding of several proteins. Here we demonstrate that the cyclophilins from man, pig, Neurospora crassa, Saccharomyces cerevisiae, and Escherichia coli are all active as prolyl isomerases and as catalysts of protein folding. This evolutionary conservation suggests that catalysis of prolyl peptide bond isomerization may be an important function of the cyclophilins. It could be related with de novo protein folding or be involved in regulatory processes. Catalysis of folding is very efficient in the presence of the high cellular concentrations of prolyl isomerase.

Amino Acid Isomerases↗
PubMed1991

GroE facilitates refolding of citrate synthase by suppressing aggregation.

The molecular chaperone GroE facilitates correct protein folding in vivo and in vitro. The mode of action of GroE was investigated by using refolding of citrate synthase as a model system. In vitro denaturation of this dimeric protein is almost irreversible, since the refolding polypeptide chains aggregate rapidly, as shown directly by a strong, concentration-dependent increase in light scattering. The yields of reactivated citrate synthase were strongly increased upon addition of GroE and MgATP. GroE inhibits aggregation reactions that compete with correct protein folding, as indicated by specific suppression of light scattering. GroEL rapidly forms a complex with unfolded or partially folded citrate synthase molecules. In this complex the refolding protein is protected from aggregation. Addition of GroES and ATP hydrolysis is required to release the polypeptide chain bound to GroEL and to allow further folding to its final, active state.

Animals↗
PubMed1990

Stability of recombinant Lys25-ribonuclease T1.

The conformational stability of recombinant Lys25-ribonuclease T1 has been determined by differential scanning microcalorimetry (DSC), UV-monitored thermal denaturation measurements, and isothermal Gdn.HCl unfolding studies. Although rather different extrapolation procedures are involved in calculating the Gibbs free energy of stabilization, there is fair agreement between the delta G degrees values derived from the three different experimental techniques at pH 5, theta = 25 degrees C: DSC, 46.6 +/- 2.1 kJ/mol; UV melting curves, 48.7 +/- 5 kJ/mol; Gdn.HCl transition curves, 40.8 +/- 1.5 kJ/mol. Thermal unfolding of the enzyme is a reversible process, and the ratio of the van't Hoff and calorimetric enthalpy, delta HvH/delta Hcal, is 0.97 +/- 0.06. This result strongly suggests that the unfolding equilibrium of Lys25-ribonuclease T1 is adequately described by a simple two-state model. Upon unfolding the heat capacity increases by delta Cp degrees = 5.1 +/- 0.5 kJ/(mol.K). Similar values have been found for the unfolding of other small proteins. Surprisingly, this denaturational heat capacity change practically vanishes in the presence of moderate NaCl concentrations. The molecular origin of this effect is not clear; it is not observed to the same extent in the unfolding of bovine pancreatic ribonuclease A, which was employed in control experiments. NaCl stabilizes Lys25-ribonuclease T1. The transition temperature varies with NaCl activity in a manner that suggests two limiting binding equilibria to be operative. Below approximately 0.2 M NaCl activity unfolding is associated with dissociation of about one ion, whereas above that concentration about four ions are released in the unfolding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1990

Isolation and sequence of an FK506-binding protein from N. crassa which catalyses protein folding.

Slow protein-folding reactions are accelerated by a prolyl cis/trans isomerase isolated from porcine kidney which is identical to cyclophilin, a protein that is probably the cellular receptor for the immunosuppressant cyclosporin A. Catalysis probably involves the isomerization of prolyl peptide bonds in the folding protein chains. Cyclosporin A inhibits folding catalysis by cyclophilin. Here we report the isolation, cloning, sequencing and expression of another protein with prolyl isomerase activity from Neurospora crassa which is unrelated to cyclophilin and which also catalyses slow steps in protein folding. This protein does, however, show sequence similarity to a human protein that binds to another, recently discovered immunosuppressive drug, FK506. Moreover, it shares 39% identity with the carboxy-terminal 114 residues of a cell-surface protein from the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease. Catalysis of folding by the FK506-binding protein from N. crassa is inhibited by FK506, but not by cyclosporin A. Thus, at least two different classes of conformationally active enzymes (conformases) exist that catalyse slow steps in protein folding. Both occur in a wide variety of cells and are inhibited by immunosuppressive drugs.

Amino Acid Isomerases↗
PubMed1990

Replacement of a cis proline simplifies the mechanism of ribonuclease T1 folding.

The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1. This is shown by a comparison of the wild type and a designed mutant protein where Ser54 and Pro55 were replaced by Gly54 and Asn55, respectively. This mutation leaves the thermal stability of the protein almost unchanged; however, in the absence of Pro55 one of the two slow phases in folding is abolished and the kinetic mechanism of refolding is dramatically simplified.

Aspergillus oryzae↗
PubMed1990

Folding of ribonuclease T1. 1. Existence of multiple unfolded states created by proline isomerization.

It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein molecules with defined sequence variations. Here we present a preliminary characterization of the folding kinetics of ribonuclease T1. Its unfolding and refolding reactions are reversible, which is shown by the quantitative recovery of the catalytic activity after an unfolding/refolding cycle. Refolding is a complex process, where native protein is formed on three distinguishable pathways. There are 3.5% fast-folding molecules, which refold within the millisecond time range, and 96.5% slow-folding species, which regain the native state in the time range of minutes to hours. These slow-folding molecules give rise to two major, parallel refolding reactions. The mixture of fast- and slow-folding molecules is produced slowly after unfolding by chain equilibration reactions that show properties of proline isomerization. We conclude that part of the kinetic complexity of RNase T1 folding can be explained on the basis of the proline model for protein folding. This is supported by the finding that the slow refolding reactions of this protein are accelerated in the presence of the enzyme prolyl isomerase. However, several properties of ribonuclease T1 refolding, such as the dependence of the relative amplitudes on the probes, used to follow folding, are not readily explained by a simple proline model.

Endoribonucleases↗
PubMed1990

Folding of ribonuclease T1. 2. Kinetic models for the folding and unfolding reactions.

The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are limited in rate by the reisomerization of incorrect proline isomers. The transient folding intermediates were also detected by unfolding assays, which make use of the reduced stability of folding intermediates relative to that of the native protein. The results of this and the preceding paper [Kiefhaber et al. (1990) Biochemistry (preceding paper in this issue)] were used to propose kinetic models for the unfolding and refolding of ribonuclease T1. The unfolding mechanism is based on the assumption that, after the structural unfolding step, the slow isomerizations of two X-Pro peptide bonds occur independently of each other in the denatured protein. At equilibrium a small amount of fast-folding species coexists with three slow-folding species: two with one incorrect proline isomer each and another, dominant species with both these prolines in the incorrect isomeric state. In the mechanism for refolding we assume that all slow-folding molecules can rapidly regain most of the secondary and part of the tertiary structure early in folding. Reisomerizations of incorrect proline peptide bonds constitute the slow, rate-limiting steps of refolding. A peculiar feature of the kinetic model for refolding is that the major unfolded species with two incorrect proline isomers can enter two alternative folding pathways, depending on which of the two reisomerizes first. The relative rates of reisomerization of the respective proline peptide bonds at the stage of the rapidly formed intermediate determine the choice of pathway. It is changed in the presence of prolyl isomerase, because this enzyme catalyzes these two isomerizations with different efficiency and consequently leads to a shift from the very slow to the intermediate refolding pathway.

Circular Dichroism↗
PubMed1990

Role of two proline-containing turns in the folding of porcine ribonuclease.

Unfolded ribonuclease (RNase) from porcine pancreas consists of a mixture of fast and slow-refolding species. The equilibrium distribution of these species differs strongly from other homologous RNases, because an additional proline residue is present at position 115 of the porcine protein. The major slow-folding species of porcine RNase contains incorrect proline isomers at Pro93 and at Pro114-Pro115. Both positions are presumably part of beta-turn structures in the native protein, as deduced from the structure of the homologous bovine RNase A. The folding kinetics of these molecules depend strongly on the conditions used. Under unfavorable conditions (near the unfolding transition), refolding is virtually blocked by the presence of the incorrect proline peptide bonds and partially folded intermediates with incorrect isomers could not be detected. As a consequence, folding is very slow under such conditions and the re-isomerization of Pro114-Pro115 is the first and rate-limiting step of folding. Under strongly native conditions (such as in the presence of ammonium sulfate), refolding is much faster. A largely folded intermediate accumulates with the turns around Pro93 and Pro114-Pro115 still in the non-native conformation. These results suggest that incorrect proline isomers strongly influence protein folding and that, under favorable conditions, the polypeptide chain can fold with two beta-turns locked into a non-native conformation. We conclude, therefore, that early formation of correct turn structure is not necessarily required for protein folding. However, the presence of incorrect turns, locked-in by non-native proline isomers, strongly decreases the rate of refolding. Alternative pathways of folding exist. The choice of pathway depends on the number and distribution of incorrect proline isomers and on the folding conditions.

Ammonium Sulfate↗
PubMed1989

Cyclophilin and peptidyl-prolyl cis-trans isomerase are probably identical proteins.

The enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was recently discovered in mammalian tissues and purified from porcine kidney. It catalyses the slow cis-trans isomerization of proline peptide (Xaa-Pro) bonds in oligopeptides and accelerates slow, rate-limiting steps in the folding of several proteins. Here, we report the N-terminal sequence of PPIase together with further chemical and enzymatic properties. The results indicate that this enzyme is probably identical to cyclophilin, a recently discovered mammalian protein which binds tightly to cyclosporin A (CsA). Cyclophilin is thought to be linked to the immunosuppressive action of CsA. The first 38 amino-acid residues of porcine PPIase and of bovine cyclophilin are identical and the two proteins both have a relative molecular mass of about 17,000 (ref. 7). The catalysis of prolyl isomerization in oligopeptides and of protein folding by PPIase are strongly inhibited in the presence of low levels of CsA. The activities of both PPIase and cyclophilin depend on a single sulphydryl group. At present it is unknown whether the inhibition of prolyl isomerase activity is related with the immunosuppressive action of CsA.

Amino Acid Isomerases↗
PubMed1989

Influence of incremental preoperative risk factors on the perioperative outcome of patients undergoing emergency versus urgent coronary artery bypass grafting.

A retrospective analysis of 127 patients with impending myocardial infarction undergoing coronary artery bypass grafting was performed to evaluate incremental risk factors associated with perioperative mortality and morbidity. Fifty-four patients (group 1) were operated upon as emergencies within 24 h and 73 patients underwent urgent coronary revascularization within a mean of 3.4 days (group II) after admission. The incidence of non-transmural myocardial infarctions (NTMI), haemodynamic parameters, the number of diseased vessels and the incidence of a preceding percutaneous coronary dilatation (PTCA) were not statistically different between the groups. The overall perioperative mortality was 8.7% (16.7% group I, 2.7% group II). Major non-fatal complications were frequent in the surviving collective including low cardiac output in 14 patients (12.1%) and transmural or subendocardial perioperative infarction in 12 patients (10.3%). Perioperative mortality was associated with reduced left ventricular myocardial function (P less than 0.001), operation within 24 hr after onset of anginal symptoms (P less than 0.001) or subendocardial infarction (P less than 0.025) in the 4 weeks before operation. Perioperative mortality was independent of the degree of coronary stenosis, number of distal anastomoses or performance of a coronary endarterectomy. Of the patients, 90.5% (87.5% of group I and 92.3% of group II) included in a mean follow-up of 16.8 months (range 5-27 months) were graded into Canadian Heart Functional Class I. Successful coronary surgery for acute myocardial ischaemia results in excellent late functional recovery. The major risk factors for fatal perioperative outcome are reduced left ventricular function and the necessity of every early surgical intervention.

Angioplasty, Balloon, Coronary↗