PubMed Health⌕ Search

Biomedical subjects

R Jerala

Publications and source records attributed to R Jerala.

15 recordsLinked to original sources
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↗
PubMed1992

Simultaneous synthesis of degenerate oligonucleotides of variable length.

Most mutagenic studies have emphasized the exchange of residues but disregarded the variation in length as the other aspect of protein variability. In this report a novel strategy to simultaneously synthesize degenerate mutagenic oligonucleotides of variable length is reported. Synthesis is done on a normal automated DNA synthesizer with modifications only in the program. Product of such synthesis can be used as a mutagenic oligonucleotide for construction of mutant proteins with variable length of inserts.

Base Sequence↗
PubMed1991

Folding studies of the cysteine proteinase inhibitor--human stefin A.

Reversible GuHCl denaturation of human stefin A (25 degrees C, pH 8) was monitored by the tyrosine fluorescence, by circular dichroism in the near UV and by circular dichroism in the far UV. In each case a midpoint of 2.8 +/- 0.1 M GuHCl was obtained, demonstrating the cooperativity of the denaturation. Kinetics of the slow folding on diluting the protein from the GuHCl denatured state, was also measured by the three spectroscopic probes (10 degrees C, pH 8). Results conform to a sequential mechanism. Denaturant concentration and temperature dependence of the slow folding were measured by fluorescence. From a linear Arrhenius plot the Ea of 100 +/- 5 kJ/mol was read. 'Double mixing' experiments revealed a slow reaction going on in the unfolded state which influenced the amplitude of the fluorescence changes. 'Double mixing' experiments performed by FPLC have shown that the folding itself, i.e., the formation of a compact state, was not dependent on the time spent under unfolding conditions.

Chromatography, High Pressure Liquid↗
PubMed1991

Deletion of the carboxy terminal part of stefin B does not have a major effect for binding to papain.

Determination of crystal structures of chicken cystatin and human stefin B complexed with papain revealed a novel model of protease inhibition and also structural differences between two cysteine proteinase inhibitor (CPI) families. According to the 3D alignment, stefins have an extension of 9 amino acids on their carboxy terminus in comparison with cystatins. The extension was not expected to make a major contribution to interaction with the enzyme. A deletion mutant of stefin B, corresponding in length to the carboxy terminal sequence of chicken cystatin, was constructed by the use of polymerase chain reaction (PCR). This (C3S, delta 89-98) human stefin B, 10 amino acids shorter, inhibited papain with a Ki of 0.012 nM which is comparable to the Ki of 0.03 nM for authentic, nondeleted recombinant stefin B. This finding thus confirms the tertiary structure-based alignment.

Amino Acid Sequence↗
PubMed1990

The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.

A stoichiometric complex of human stefin B and carboxymethylated papain has been crystallized in a trigonal crystal form. Data to 2.37 A resolution were collected using the area detector diffractometer FAST. The crystal structure of the complex has been solved by Patterson search techniques using papain as search model. Starting from the structure of chicken cystatin, the stefin structure was elucidated through cycles of model building and crystallographic refinement. The current crystallographic R factor is 0.19. Like cystatin, the stefin molecule consists of a five stranded beta-sheet wrapped around a five turn alpha-helix, but with an additional carboxy terminal strand running along the convex side of the sheet. Topological equivalence of stefin and cystatin reveal the previous sequence alignment to be incorrect in part, through deletion of the intermediate helix. The conserved residues form a tripartite wedge, which slots into the papain active site as proposed through consideration of the tertiary structures of the individual components (Bode et al., 1988). The main interactions are provided by the amino terminal 'trunk' (occupying the 'unprimed' subsites of the enzyme), and by the first hairpin loop, containing the highly conserved QVVAG sequence, with minor contributions from the second hairpin loop. The carboxyl terminus of stefin provides an additional interaction region with respect to cystatin. The interaction is dominated by hydrophobic contacts. Inhibition by the cysteine proteinase inhibitors is fundamentally different to that observed for the serine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1990

A new inhibitor of plasmin and trypsin from porcine leukocytes.

A new intracellular inhibitor of plasmin and trypsin was isolated from porcine leukocytes by ion exchange chromatography and affinity chromatography. In dodecyl sulphate gel electrophoresis a single protein band with an apparent molecular mass of 15 kDa was found under reducing conditions. On isoelectric focusing three protein bands with isoelectric points between pH 4.0 and 4.5 were found. The association rate constants and the inhibition constants were determined for porcine plasmin and bovine trypsin. The inhibitor shows no immunologic cross-reactivity with any of the tested leukocyte inhibitors. On the basis of its N-terminal amino-acid sequence a great degree of similarity to Kunitz-type inhibitors was observed.

Amino Acid Sequence↗
PubMed1990

Mutations in the QVVAG region of the cysteine proteinase inhibitor stefin B.

Variants of human stefin B were constructed by cassette mutagenesis. Val47 as the constituent of highly conserved QVVAG sequence was substituted by hydrophobic amino acids of increasing size - Ala, Ile and Phe. Recombinant proteins were expressed in E. coli and Ki values for papain were determined. Substitutions did not cause a major change in Ki value and we conclude that the interaction with the proteinases is not the reason for the conservation of the pentapeptide.

Base Sequence↗
PubMed1989

Primary structure of a new cysteine proteinase inhibitor from pig leucocytes.

The primary structure of a pig leucocyte cysteine proteinase inhibitor, also called cathelin, was determined. The sequence was obtained from analyses of peptides isolated from the chymotryptic, endoproteinase Lys-C and protease V8 digests, and by analysis of the peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The inhibitor consists of 96 residues. The N-terminal residue of the inhibitor is pyrrolidone-carboxylic acid. The amino acid sequence of cathelin suggests the appearance of a new family of cysteine proteinase inhibitors.

Amino Acid Sequence↗
PubMed1988

Cloning a synthetic gene for human stefin B and its expression in E. coli.

A gene coding for human stefin B was synthesized by the solid-phase phosphite method and cloned in the pUC8 cloning vector. The insert with the verified DNA sequence was subcloned into two expression vectors and expressed in E. coli as a fusion protein with beta-galactosidase and as a native protein. The CNBr cleaved fusion protein and the native recombinant stefin B were inhibitory to papain and reacted with antibodies against human stefin B.

Amino Acid Sequence↗
PubMed1988

Regen: program for designing gene assembly.

One of the main problems in constructing synthetic genes is the incorrect hybridisation between the oligonucleotides. The problem is resolved if the sequence uniquely defines the position of the oligonucleotide in the assembled gene. This can be accomplished through the wise partition of dsDNA sequence in the fragments. We describe a program for use in designing such gene assembly. For a given DNA sequence and the approximate location of oligonucleotide boundary it generates all sets of protruding ends that share the smallest homology.

Algorithms↗