Biomedical subjects

A J Schulze

Publications and source records attributed to A J Schulze.

12 recordsLinked to original sources

PubMed1997

Conformational dynamics and kinetics of peptide antagonist interactions with interleukin-1 receptor. Fluorescence studies using the NBD-labelled peptide AF12415.

Interleukin-1 plays a key role in the inflammatory response provoked by various disease states and inhibition of its action can bring therapeutic benefits. Steady-state and time-resolved studies of the intrinsic tryptophan fluorescence of the free soluble Type I form of interleukin-1 receptor (IL-1R) reveal that the rotational motions of the three major domains are strongly associated. Bound peptide antagonists are buried in hydrophobic regions, but a flexible association permits access to species from the aqueous phase. Ligand binding does not lead to rigidification of the receptor structure. The kinetics and mechanism of complex formation and dissociation, involving IL-1R with receptor antagonist protein (IL-1ra) and with peptides AF11733 (15 aa) and AF10961 (21 aa) were determined with the aid of peptide AF12415 (15 aa) labeled at its N-terminus by the NBD fluorophore, which exhibits a five-fold increase in emission intensity at 540 nm on binding of the peptide to IL-1R. The magnitude of the ON rate constant, typically 1 x 10(6) M-1 s-1, implies the existence of an intermediate 'encounter complex' involving interactions of low specificity. Readjustments of the initial encounter complex leads to a final complex where very specific interactions dominate. The first-order rate constant for this latter process is the most sensitive indicator of the true peptide affinity for the receptor binding site, and thus provides a better criterion than the apparent OFF rate (typically 2 x 10(-3) s-1) for discrimination of peptide antagonists.

Amino Acid Sequence↗

PubMed1996

A spectroscopic study of the structures of latent, active and reactive-center-cleaved type-1 plasminogen-activator inhibitor.

Type-1 plasminogen-activator inhibitor (PAI-1) was studied by Fourier-transform infrared spectroscopy, far-ultraviolet CD spectroscopy, and fluorescence-emission spectroscopy, with the aim to obtain structural information about its active form. The spectra of latent, active and reactive-center-cleaved forms of PAI-1 produced by HT-1080 cells were different. While the cleaved and the latent forms were similar with regard to their beta-structure content, comparison of the spectra of these forms with the spectra of active PAI-1 suggested a much higher degree of unordered structure for the active form compared with the latent and reactive-center-cleaved forms than previously assumed. We discuss our results with reference to the known three-dimensional X-ray structures of latent PAI-1, of reactive-center-cleaved serpins, including reactive-center-cleaved PAI-1, and of intact serpins, and with reference to previous results on the differences in the affinity of mAbs for the different PAI-1 forms. We interpret our results in favor of a global rearrangement of secondary structure during latency transition and reactive-center cleavage in PAI-1, not only involving the reactive-center loop and parts of beta-sheets A and C, but also the "rear' side of the molecule, such as helices H and G. Thus, we suggest flexibility in serpin structural elements that were previously regarded as rigid.

Binding Sites↗

PubMed1996

Transient non-native interactions in early folding intermediates do not influence the folding kinetics of Escherichia coli tryptophan synthase beta 2 subunits.

Local structures formed in early intermediates are thought to play a key role in orientating the protein folding pathway. Here, we test whether non-native interactions formed by eight N-terminal residues in early folding intermediates of tryptophan synthase beta chains [Navon, A., Schulze, A. J., Guillou, Y., Zylinksii, C. A., Baleux, F., Expert-Bezançon, N., Friguet, B., Djavadi-Ohaniance, L. & Goldberg, M. E. (1995) J. Biol. Chem. 270, 4255-4261] influence the folding kinetics. The kinetics of in vitro renaturation of wild-type beta chains and truncated beta chains lacking residues 2-9 were compared. Each step analyzed (molten globule formation, tryptophan shielding, closing up of distant residues, and complete renaturation) occurred with similar kinetics in the normal and truncated proteins. Thus, non-native interactions formed early during the folding of beta chains seem to influence neither the rate and efficiency of the complete renaturation, nor the kinetics of the early folding steps, which suggests that such non-native early intermediates are poorly stable and highly dynamic. This observation is consistent with the current view that productive folding should avoid the formation of stable intermediates.

Anilino Naphthalenesulfonates↗

PubMed1995

Importance of residues 2-9 in the immunoreactivity, subunit interactions, and activity of the beta 2 subunit of Escherichia coli tryptophan synthase.

The epitope recognized by a monoclonal antibody (mAb19) directed against the beta 2 subunit of Escherichia coli tryptophan synthase was found to be carried by residues 2-9 of the beta chain. The affinities of mAb19 for peptides of different lengths containing the 2-9 sequence were close to 0.6 x 10(9) M-1, the affinity of mAb19 for native beta 2. In view of these results, a model is proposed to account for the kinetics of appearance of the epitope during in vitro renaturation of beta 2 (Murry-Brelier, A., and Goldberg, M.E. (1988) Biochemistry 27, 7633-7640). A mutant producing beta chains lacking residues 1-9 (beta delta 1-9) was prepared. The beta delta 1-9 protein was able to fold into a heat stable homodimer resembling wild type beta 2. Isolated beta delta 1-9 had no detectable enzymatic activity. It could bind alpha chains extremely weakly and be slightly activated. In the presence of the 1-9 peptide, the beta delta 1-9 protein could bind alpha chains much more strongly and generate a 50% active enzyme. Thus, although having little role in the overall folding and stability of the protein, the 1-9 sequence of the beta chain appears strongly involved in the alpha-beta interactions and in the enzymatic activity.

Antibodies, Monoclonal↗

PubMed1995

Divining the serpin inhibition mechanism: a suicide substrate 'springe'?

The most important of diverse serpin functions is serine-protease inhibition. In contrast to the 'standard-mechanism' inhibitors, inhibitory serpins use a mechanism that involves unusual flexibility, and cofactor and receptor interactions. The principal feature is a refolding step, during which a disordered or helical strand is inserted into the center of a beta sheet. This transition, which is essential for inhibition, can be induced by heating, proteolytic cleavage of the serpin, or complexation with the proteinase target; analogous transitions can be induced by peptide complexation or aggregation. Although it is difficult to determine the details of this mechanism, information derived from crystal structures and other experiments has stimulated drug design efforts with wide-ranging potential applications.

Crystallography, X-Ray↗

PubMed1994

Production, inhibitory activity, folding and conformational analysis of an N-terminal and an internal deletion variant of chicken cystatin.

Two deletion variants of chicken cystatin were produced after cassette mutagenesis of the recombinant Arg-Glu-Phe-[Met1, Ile29, Leu89]-chicken egg white cystatin gene in Escherichia coli. The variant des-Ser1-Pro11-[Ala12, Glu13, Phe14, Met15, Ile29, Leu89]-chicken cystatin (N-del 2) and the variant Arg-Glu-Phe-[Met1, Ile29]-des-Cys71-Met89-chicken cystatin (del-helix II) were purified and characterized by inhibition kinetics, far-ultraviolet-CD and fluorescence spectroscopy, and their folding in guanidine hydrochloride (Gdn/HCl) was studied. The del-helix II variant, shortened by 19 amino acids, is a basic, stefin-like mini-cystatin with one disulfide bridge. Its inhibitory properties are identical to chicken cystatin and its stability against Gdn/HCl is similar. The folding of the del-helix II variant corresponds best to a single step process. In contrast to this, the reversible folding of natural and recombinant chicken cystatin is more complex when recorded by either tryptophan fluorescence or far-ultraviolet-CD. With increasing Gdn/HCl concentration, a stabilization of secondary-structural elements is initially observed, followed by unfolding with minor but distinct intermediate states. The N-del 2 variant has a neutral pI and shows folding behaviour very similar to natural and recombinant chicken cystatin. However its inhibition constants with papain, actinidin and cathepsin B and L are 1000-100,000-fold higher than those obtained with natural and recombinant chicken cystatin.

Amino Acid Sequence↗

PubMed1994

Structural aspects of serpin inhibition.

The essential roles of proteins of the serpin family in many physiological processes, along with new discoveries of their unique folding properties, have attracted intense interest in recent years. Many serpins display unusual mobile behavior attributed to rearrangements of alpha-helical or beta-sheet domains, whereby large scale transitions accompany a variety of functions, including inactivation. This unusual behavior was first recognized with the X-ray structure of modified alpha 1-proteinase inhibitor. Subsequent experiments, including new X-ray structures, have revealed a surprising variety of conformations which are functionally important but only partially understood. We review here experimental evidence for conformations relevant to the serpin inhibitory mechanism.

Crystallography, X-Ray↗

PubMed1994

Expression of alpha 1-proteinase inhibitor in Escherichia coli: effects of single amino acid substitutions in the active site loop on aggregate formation.

Overproduction of eukaryotic proteins in microorganisms often leads to the formation of insoluble protein aggregates which accumulate as intracellular inclusion bodies. alpha 1-Proteinase inhibitor (alpha 1-PI) when produced as a cytoplasmic protein in Escherichia coli (E. coli) forms inclusion bodies containing the majority of the inhibitor in an inactive form. Several variants of alpha 1-PI with single amino acid substitutions within their active site loop (amino acids 345-358) were produced in a bioreactor showing that substitution of Met351 with Glu resulted in significantly reduced aggregate formation compared to the other variants and to wild-type protein. In addition, this variant proved to be fully functional as a proteinase inhibitor. Based on these findings and on results of previous structural studies a mechanism for aggregate formation during expression of alpha 1-PI is suggested.

Binding Sites↗

PubMed1993

Serpin structures.

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Amino Acid Sequence↗

PubMed1992

Evidence for the extent of insertion of the active site loop of intact alpha 1 proteinase inhibitor in beta-sheet A.

The extent of insertion of beta-strand s4A into sheet A in intact serpin alpha 1-proteinase inhibitor (alpha 1PI has been probed by peptide annealing experiments [Schulze et al. (1990) Eur. J. Biochem. 194, 51-56]. Twelve synthetic peptides of systematically varied length corresponding in sequence to the unprimed (N-terminal) side of the active site loop were complexed with alpha 1PI. The complexes were then characterized by circular dichroism spectroscopy and tested for inhibitory activity. Four peptides formed complexes which retained inhibitory activity, one of which was nearly as effective as the native protein. Comparison with the three dimensional structures of cleaved alpha 1PI [Löbermann et al. (1984) J. Mol. Biol. 177, 531-556] and plakalbumin [Wright et al. (1990) J. Mol. Biol. 213, 513-528] supports a model in which alpha 1PI requires the insertion of a single residue, Thr345, into sheet A for activity.

Amino Acid Sequence↗

PubMed1991

Inhibitory activity and conformational transition of alpha 1-proteinase inhibitor variants.

Several variants of alpha 1-proteinase inhibitor (alpha 1-PI) were investigated by spectroscopic methods and characterized according to their inhibitory activity. Replacement of Thr345 (P14) with Arg in alpha 1-PI containing an Arg residue in position 358 (yielding [Thr345----Arg, Met358----Arg]alpha 1-PI) results in complete loss of its inhibitory activity against human alpha-thrombin; whereas an exchange of residue Met351 (P8) by Glu [( Met351----Glu, Met358----Arg]alpha 1-PI) does not alter activity. [Thr345----Arg, Met358----Arg]alpha 1-PI is rapidly cleaved by thrombin, while [Met358----Arg]alpha 1-PI and [Met351----Glu, Met358----Arg]alpha 1-PI form stable proteinase-inhibitor complexes. The stability of [Thr345----Arg, Met358----Arg]alpha 1-PI against guanidinium chloride denaturation is significantly enhanced compared to wild-type alpha 1-PI, and does not change after cleavage, resembling ovalbumin, a serpin with no inhibitory activity, from which the Thr345----Arg amino acid exchange had been derived. [Met351----Glu, Met358----Arg]alpha 1-PI and [Met358----Arg]alpha 1-PI resemble the wild-type protein in this respect. The CD spectra of intact and cleaved alpha 1-PI variants do not compare well with the wild-type protein, probably reflecting local structural differences. Insertion of a synthetic peptide, which corresponds to residues Thr345----Met358 of human alpha 1-PI, leads to the formation of binary complexes with all variants having the characteristic features of the binary complex between peptide and wild-type protein.

Amino Acid Sequence↗

PubMed1990

Structural transition of alpha 1-antitrypsin by a peptide sequentially similar to beta-strand s4A.

Crystal structure studies have shown that cleaved and intact serpins differ essentially in the topology of beta-sheet A. This is five-stranded in the intact molecules and six-stranded after cleavage by insertion of strand s4A whose C-terminus has become free [Löbermann, H., Tokuoka, R., Deisenhofer, J. & Huber, R. (1984) J. Mol. Biol. 177, 531-556; Wright, T. H., Qian, H. X. & Huber, R. (1990) J. Mol. Biol. 213, 513-528]. The structural transition is accompanied by changes in spectral properties and an increase in thermal stability. We show here that an N alpha-acetyl-tetradecapeptide with the amino acid sequence of strand s4A, residues 345-358 of human alpha 1-antitrypsin, associates with intact alpha 1-antitrypsin and forms a stoichiometric complex with properties very similar to cleaved alpha 1-antitrypsin. Complex generation has the characteristics of a folding process.

Amino Acid Sequence↗