Biomedical subjects

R Jerala

Publications and source records attributed to R Jerala.

At least 19 recordsLinked to original sources

PubMed1998

On the mechanism of human stefin B folding: II. Folding from GuHCl unfolded, TFE denatured, acid denatured, and acid intermediate states.

It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Zerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8+/-0.2, an acid molten globule intermediate I1 (pH 3.3+/-0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3+/-0.1, 0.42 M NaCl), and the TFE state (pH 3.3+/-0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2.

Anilino Naphthalenesulfonates↗

PubMed1998

On the mechanism of human stefin B folding: I. Comparison to homologous stefin A. Influence of pH and trifluoroethanol on the fast and slow folding phases.

The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60+/-5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved "burst" phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6s) detected by all the spectroscopic probes, which occurred subsequent to an initial "burst" phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed.

Cystatin A↗

PubMed1998

pH-induced conformational transitions of the propeptide of human cathepsin L. A role for a molten globule state in zymogen activation.

Synthesis of proteases as inactive zymogens is a very important mechanism for the regulation of their activity. For lysosomal proteases proteolytic cleavage of the propeptide is triggered by the acidic pH. By using fluorescence, circular dichroism, and NMR spectroscopy, we show that upon decreasing the pH from 6.5 to 3 the propeptide of cathepsin L loses most of the tertiary structure, but almost none of the secondary structure is lost. Another partially structured intermediate, prone to aggregation, was identified between pH 6.5 and 4. The conformation, populated below pH 4, where the activation of cathepsin L occurs, is not completely unfolded and has the properties of molten globule, including characteristic binding of the 1-anilinonaphthalene-8-sulfonic acid. This pH unfolding of the propeptide parallels a decrease of its affinity for cathepsin L and suggests the mechanism for the acidic zymogen activation. Addition of anionic polysaccharides that activate cathepsin L already at pH 5.5 unfolds the tertiary structure of the propeptide at this pH. Propeptide of human cathepsin L which is able to fold independently represents an evolutionary intermediate in the emergence of novel inhibitors originating from the enzyme proregions.

Cathepsin L↗

PubMed1997

Characterization of the equilibrium intermediates in acid denaturation of human stefin B.

Acid-induced denaturation of recombinant human stefin B was followed using circular dichroism (CD) and fluorimetry. By comparing different spectroscopic probes, a number of equilibrium intermediates were detected. In pH denaturation at very low salt concentration (0.03 M NaCl) four states can be distinguished: N - I(N) - I1 - U, where N is the native state, I(N) is a native-like intermediate, I1 is an acid intermediate state with properties of a molten globule and U is the unfolded state. State 1, exhibits no near-ultraviolet CD but has some residual far-ultraviolet CD. It differs from U in its ability to increase fluorescence of 1-anilino-naphthalene 8-sulfonate (ANS). In 0.42 M salt, the pH denaturation is three-state between the dimeric native state N2 and intermediates I(N2) and I2, which are also dimeric according to size-exclusion chromatography. The acid intermediate I2 is more structured than I1: it binds ANS to a lower extent an I1, its Tyr residues are protected from the solvent, it shows some near-ultraviolet CD and its far-ultraviolet CD is even more intense than that for the native state. 1H-NMR spectra confirmed the overall structural features of the acid intermediates. To obtain the enthalpies of unfolding, microcalorimetric measurements were performed under conditions where the acid intermediates are maximally populated (18 degrees C): state I(N) from pH 5.0 to 4.6, 0.03 M salt: state I1 below pH 3.8, 0.42 M salt; and state I1 in equilibrium with I(N) at pH 4.05, 0.03 M salt. Enthalpies of unfolding for states I(N) and I1 were comparable to those of the native state. The enthalpy of unfolding for state I1 could not be determined.

Anilino Naphthalenesulfonates↗

PubMed1996

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: presence of an amino-terminal helix in solution.

1H, 15N and 13C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007-1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G. H., Kaptein, R. and Verheij, H.M. (1995) Nature Strct. Biol., 2, 402-406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned amino-terminal helix in the absence of substrate. Therefore, there is a clear difference between the conformation group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.

Amino Acid Sequence↗

PubMed1996

Simulation of the gel-fluid transition in a membrane composed of lipids with two connected acyl chains: application of a dimer-move step.

Phospholipids have been treated as dimers on a hexagonal lattice, and a move has been introduced that allows the dimers to move and change their orientation on the lattice. Simulations have been performed in which phospholipid chains have been treated as being either independent or infinitely coupled thermodynamically with regard to their conformational state. Both types of simulation have reproduced well experimental heat-capacity curves of dipalmitoyl phosphatidylcholine small unilamellar vesicles. Apart from a different gel-fluid interaction parameter and a different number of unlike nearest-neighbor contacts, most of the averages and thermodynamic quantities were essentially the same in the two types of simulation. These results indicate that the transition is not first order and validate those of previous Monte Carlo simulations that have neglected the dimeric nature of phospholipids in the sense that they show that for the thermotropic transition the approximation of phospholipids as monomers is valid.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1995

The three-dimensional solution structure of human stefin A.

The three-dimensional solution structure of recombinant human stefin A has been determined by a simulated annealing protocol using a total of 1113 distance and angle constraints obtained from 1H and 15N HMR spectroscopy. The solution structure is represented by a family of 17 conformers with an average root-mean-square deviation relative to the mean structure of 0.44 A for backbone atoms and 0.94 A for all heavy atoms for the main body of the structure. The protein has a well-defined global fold consisting of five anti-parallel beta-strands wrapped around a central five-turn alpha-helix. There is considerable similarity between the structural features of free stefin A in solution and the X-ray structure of the homologous protein stefin B in its complex with papain, but there are also some important differences in the regions which are fundamental to proteinase binding. The differences consist primarily of two regions of high conformational heterogeneity in free stefin A which correspond in stefin B to two of the components of the tripartite wedge that docks into the active site of the target proteinase. These regions, which are shown to be mobile in solution, are the five N-terminal residues and the second binding loop. In the bound conformation of stefin B they form a turn and a short helix, respectively.

Amino Acid Sequence↗

PubMed1994

Compactness of the molten globule in comparison to unfolded states as observed by size-exclusion chromatography.

The volumes of elution of denatured states of four proteins at high urea (8 M) and ethylurea (6 M) concentration were determined. They were found equally unfolded in both solvents. The volumes of elution of the unfolded states were compared to those of the native states and of some molten globule intermediates. It has been shown that the protein proteinase inhibitor stefin B, exhibits 'molten globule'-like properties on acid denaturation. The high salt acidic intermediate (a molten globule) as well as the native state of stefin B eluted as dimers, at 18 degrees C. On thermal denaturation above 42 degrees C, the intermediate dissociated into compact monomers. The more stable stefin A, which is monomeric and does not transform into molten globule intermediates under similar perturbing conditions, was always used for comparison. The states of both, stefin A and B in 50% methanol were found to be monomeric and of native-like compactness.

Chromatography, Gel↗

PubMed1994

Structural characterisation of human stefin A in solution and implications for binding to cysteine proteinases.

Stefin A is a member of the cystatin superfamily of proteins which are tight and reversibly binding inhibitors of the papain-like cysteine proteinases. The 1H-NMR and 15N-NMR resonances of human stefin A have been sequentially assigned using two-dimensional homonuclear and heteronuclear NMR techniques in conjunction with three-dimensional heteronuclear methods. Characteristic sequential and medium range NOE contacts, J constants and hydrogen exchange data have been used to identify the secondary structural elements of the protein which consists of five anti-parallel beta-strands and a single alpha-helix. There is much similarity between the secondary structural features of stefin A and the homologous protein stefin B in its complex with papain [Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B. & Turk, V. (1990) EMBO. J. 9. 1939-1947] but also some important differences in regions which are fundamental to the binding event. The principal difference is the presence of two conformationally unrestricted regions in stefin A that form two of the components of the tripartite wedge which docks into the active site of the target proteinase. Specifically, these regions are the five N-terminal residues and the second binding loop, which form a turn and a short helix respectively, in the bound conformation of stefin B.

Amino Acid Sequence↗

PubMed1994

Elongation on the amino-terminal part of stefin B decreases inhibition of cathepsin H.

Two mutants of the cysteine proteinase inhibitor, stefin B, were prepared by ligating the amino-terminal region from cystatin C and kininogen, members of two other families of cystatin superfamily. The mutant proteins were expressed in Escherichia coli and purified to homogeneity. Inhibition and kinetic constants were determined for authentic and mutated stefins against the four different cysteine proteinases, papain and human cathepsins B, L and H. Inhibition of both amino-terminal elongated stefin B mutants was decreased particularly for cathepsin H. A model of the tertiary structure of cathepsin H and its complex with stefin B was constructed. The framework for the model of cathepsin H consisted of structurally conserved regions from tertiary structures of three cysteine proteinases. Variable regions were selected from fragments of other proteins from the protein data base. We suggest that reduced binding of stefins with elongated amino termini is caused by the mini chain of cathepsin H which is probably in close proximity to the amino termini in the complexes. This mini chain is bridged to Cys214 and has already been proposed to be responsible for the aminopeptidase activity of cathepsin H. We conclude that the amino-terminal region of stefin B plays an important role in determining the strength of inhibition of cathepsin H.

Amino Acid Sequence↗

PubMed1994

Improved expression and evaluation of polyethyleneimine precipitation in isolation of recombinant cysteine proteinase inhibitor stefin B.

Synthetic gene coding for human cysteine proteinase inhibitor stefin B was expressed in Escherichia coli by the use of pKP1500 plasmid-containing tac promotor and temperature-sensitive origin of replication, ensuring high plasmid copy number. Several parameters were varied in order to maximize the yield of inhibitory active protein: distance between RBS and initiator codon, temperature of fermentation, and conditions of fermentation. Production of stefin B was markedly improved by setting the RBS to ATG codon distance to 10 nt and with fermentation conditions that increased yield of biomass. The isolation procedure was modified by including precipitation with polyethyleneimine that removed contaminants such as nucleic acids and most bacterial (predominantly acidic) proteins. Precipitation itself produced more than 80% pure recombinant inhibitor, which was purified to homogeneity by a single chromatographic step. Isolated protein had the same inhibitory properties as authentic inhibitor.

Base Sequence↗

PubMed1994

Structural basis for the difference in thermodynamic properties between the two cysteine proteinase inhibitors human stefins A and B.

Homology modelling has been used to model stefin A based on the X-ray structure of stefin B. Several models have been produced by interactive modelling or positioning of the side chains by Monte Carlo procedure with simulated annealing. The quality of models was evaluated by calculation of the free energy of hydration, 3D-1D potential or buried area of surface accessibility. Stefin A is a thermostable protein, exhibiting a two-state denaturation, while stefin B denatures at a 40 degrees C lower temperature and forms a stable molten globule intermediate under mild denaturing conditions. From the tertiary structures, thermodynamic functions were predicted, conforming closely to the experimental calorimetric results. Polar and apolar buried areas of surface accessibility were obtained by structural deconvolution of the thermograms. It is suggested that the basic difference between the stefins is the domination of hydrophobic interaction in the stabilization of stefin B, which is due to its non-specific nature leading to the formation of a molten globule intermediate. Modelling of stefin A predicts increased numbers of hydrogen bonds which stabilize it and increase the cooperativity of its denaturation.

Amino Acid Sequence↗

PubMed1993

The primary structure of inhibitor of cysteine proteinases from potato.

The complete amino acid sequence of the cysteine proteinase inhibitor from potato tubers was determined. The inhibitor is a single-chain protein having 180 amino acid residues. Its primary structure was elucidated by automatic degradation of the intact protein and sequence analysis of peptides generated by CNBr, trypsin and glycyl endopeptidase. A search through the protein sequence database showed homology to other plant proteinase inhibitors of different specificities and non-inhibitory proteins of M(r) around 20,000. On the basis of sequence homology, prediction of secondary structure and fold compatibility, based on a 3D-1D score to the three-dimensional profile of Erythrina caffra trypsin inhibitor, we suggest that the potato cysteine proteinase inhibitor belongs to the superfamily of proteins that have the same pattern of three-dimensional structure as soybean trypsin inhibitor. This superfamily would therefore include proteins that inhibit three different classes of proteinases-serine, cysteine and aspartic proteinases.

Amino Acid Sequence↗

PubMed1993

Monoclonal antibodies to human stefin B and determination of their epitopes.

Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.

Amino Acid Sequence↗

PubMed1992

Calorimetric measurements of thermal denaturation of stefins A and B. Comparison to predicted thermodynamics of stefin-B unfolding.

Thermal denaturation of two homologous proteins, low-M(r) cysteine-proteinase inhibitors stefins A and B, has been investigated by microcalorimetry. Calorimetric enthalpies, as well as the temperatures at maximum heat capacity, were determined as a function of pH for each protein. Transitions were found reversible at all pH values examined (5.0, 6.5, 8.1) for the thermally more stable stefin A, in contrast to stefin B. Stefin B shows a sharp irreversible transition around 65 degrees C at pH 6.5 and 8.1, probably due to unfolding of a dimeric state followed by oligomerisation. At pH 5.0, both proteins exhibit a reversible transition with temperatures of half-denaturation at 50.2 degrees C and 90.8 degrees C for stefins B and A, respectively. The calorimetric enthalpies, which equal the van't Hoff enthalpies to within 10%, are 293 kJ/mol and 490 kJ/mol for stefins B and A, respectively. Using the predictive method of Ooi and Oobatake (1991) [Proc. Natl Acad. Sci. USA 88, 2859] the thermodynamic functions of unfolding were calculated for stefin B, whose three-dimensional structure has been determined. The calculated enthalpy, heat-capacity change on unfolding and the temperature of half denaturation compare well to the microcalorimetric data.

Calorimetry, Differential Scanning↗

PubMed1992

Intermediates in denaturation of a small globular protein, recombinant human stefin B.

Guanidinium HCl (GdmHCl), pH, and heat denaturation of the recombinant human stefin B, a low molecular weight protein inhibitor of cysteine proteinases, has been followed by circular dichroism. From the noncoincidence of the transitions in the near and far UV, the existence of stable intermediate states possessing few persistent tertiary interactions but most of the native-like secondary structure, was inferred. These intermediate states exist at equilibrium under various conditions, namely, state G at 1.7 M GdmHCl (pH 8, 25 degrees C), state A at pH 4 (0.6 M GdmHCl, 25 degrees C) and state T above 68 degrees C. By size exclusion chromatography, their apparent compactness was determined. The intermediate states A, T, and G were compact and are therefore classified as "molten globule" states.

Chromatography, Gel↗

PubMed1992

Conformation and fluctuations of free stefin B: a molecular dynamics study.

Molecular dynamics study was performed on the cysteine proteinase inhibitor stefin B. Structure of inhibitor from the complex with papain was used as a starting point. Amino terminal "trunk" of the inhibitor which lies extended along the cleft of the enzyme in the complex, folded onto the body of inhibitor during MD simulation, thereby reducing the total and particularly hydrophobic surface exposed to the solvent. This effect counterbalances hydrophobic contribution of the "trunk" and explains why its deletion in stefin B and related inhibitors doesn't reduce the dissociation constant. The rest of stefin B conformation is conserved together with main chain hydrogen bonds. Fluctuations of C alpha atoms resembles crystallographic B factors with exception of residues in contact with enzyme.

Chemical Phenomena↗

PubMed1992

Denaturation of stefin B by GuHCl, pH and heat; evidence for molten globule intermediates.

GuHCl, pH and thermal denaturation of the recombinant stefin B was followed by circular dichroism (CD) and size-exclusion chromatography (SEC). CD at 277 nm was taken as an indicator of integral tertiary structure and CD at 222 nm as an indicator of the secondary structure. Compactness was expressed by the volumes of elution on a SEC (Superose 12) column. Data on equilibrium denaturation were recalculated to the fractions of the native state (fN). The results have shown that equilibrium intermediates of the molten globule type exist under conditions of low pH, high temperature or medium GuHCl concentrations, namely A, T and G. Recent findings on structure and energetics of molten globule intermediates are reviewed.

Circular Dichroism↗