Disulfide bonds are known to be crucial for protein stability. To probe the contribution of each of the five disulfide bonds (C9-C31, C30-C70, C37-C63, C61-C95, and C105-C113) in bee venom phospholipase A(2) to stability, variants with deleted disulfide bonds were produced by substituting two serine residues for each pair of cysteine residues. The mutations started from the pseudo-wild-type variant (pWT) with the mutation I1A (Markert et al., Biotechnol. Bioeng. 98 (2007) 48-59). All variants were expressed in Escherichia coli, refolded from inclusion bodies and purified as pWT. The activity of the variants ranged from 12 to 82% of pWT. From the transition curves of guanidine hydrochloride-induced unfolding, the contributions of the individual disulfide bonds to conformational stability were estimated. They increased in the sequence C9-C31 <C105-C113 <C30-C70 approximate to C37-C63 < C61-C95. For two disulfide bonds (C9-C31, C105-C113) the effects were confirmed on additionally produced variants with the substitution of cysteine by alanine. Despite distinct differences in stability, all variants showed similar cooperativity in unfolding. Selected variants were also probed for proteolytic stability toward thermolysin. The removal of disulfide bonds increased the proteolytic susceptibility of the native proteins in the same way as the stability decreased. From the comparison of the results with literature data on phospholipase A(2) from bovine pancreas possessing seven disulfide bonds, it was concluded that conserved disulfide bonds in homologous proteins fulfill related functions in conformational stability. (C) 2010 Elsevier Masson SAS. All rights reserved.

Chemie Ingenieur TechnikVolume 78, Issue 9 p. 1408-1408 VortragFree Access Design zytotoxischer RNase A-Tandemenzyme als potenzielle Tumortherapeutika F. Leich, franziska.leich@biochemtech.uni-halle.de Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorJ. Köditz Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorR. Ulbrich-Hofmann Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorU. Arnold Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this author F. Leich, franziska.leich@biochemtech.uni-halle.de Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorJ. Köditz Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorR. Ulbrich-Hofmann Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this authorU. Arnold Dr., Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 HalleSearch for more papers by this author First published: 06 September 2006 https://doi.org/10.1002/cite.200650342AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume78, Issue9Special Issue: GVC/DECHEMA‐Jahrestagungen 2006 mit 24. DECHEMA‐Jahrestagung der BiotechnologenSeptember, 2006Pages 1408-1408 ReferencesRelatedInformation

Due to their ability to degrade RNA, selected members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol of target cells, where they degrade cellular RNA and cause cell death. The cytotoxic activity of most RNases, however, is abolished by the cytosolic ribonuclease inhibitor (RI). Consequently, the development of RNase derivatives with the ability to evade RI binding is a desirable goal. In this study, tandem enzymes consisting of two RNase A units that are bound covalently via a peptide linker were generated by gene duplication. As deduced from the crystal structure of the RNase A.RI complex, one RNase A unit of the tandem enzyme can still be bound by RI. The other unit, however, should remain unbound because of steric hindrance. This free RNase A unit is expected to maintain its activity and to act as a cytotoxic agent. The study of the influence of the linker sequence on the conformation and stability of these constructs revealed that tandemization has only minor effects on the activity and stability of the constructs in comparison to monomeric RNase A. Relative activity was decreased by 10-50% and the melting temperature was decreased by less than 2.5 K. Furthermore, the cytotoxic potency of the RNase A tandem enzymes was investigated. Despite an in vitro inhibition by RI, tandemization was found to endow RNase A with remarkable cytotoxic activity. While monomeric RNase A is not cytotoxic, IC(50) values of the RNase A tandem variants decreased to 70.3-12.9 microM. These findings might establish the development of a new class of chemotherapeutic agents based on pancreatic ribonucleases.

The structure of a protein molecule consists of both rigid and flexible sections to satisfy the demands for stability and catalysis. Because the flexibility of a protein segment is indispensable for a proteolytic attack, limited proteolysis is a superb tool to analyse both confined local fluctuations and global unfolding events in proteins. While the identification of the primary cleavage products allows the assignment of the flexible regions to the primary structure, the kinetics of proteolytic degradation enables differentiation between local fluctuations in the native protein molecule and the global unfolding process during denaturation. Modifications of the amino acid sequence in the concerned regions can tune proteolytic susceptibility and alter protein stability. In the present paper, we summarise our results on native-state and unfolded-state proteolysis of ribonuclease A (RNase A) and the effect of mutations in the detected flexible regions on the stability and unfolding of the RNase A molecule.

Ribonuclease A contains two exposed loop regions, around Ala20 and Asn34. Only the loop around Ala20 is sufficiently flexible even under native conditions to allow cleavage by nonspecific proteases. In contrast, the loop around Asn34 (together with the adjacent beta-sheet around Thr45) is the first region of the ribonuclease A molecule that becomes susceptible to thermolysin and trypsin under unfolding conditions. This second region therefore has been suggested to be involved in early steps of unfolding and was designated as the unfolding region of the ribonuclease A molecule. Consequently, modifications in this region should have a great impact on the unfolding and, thus, on the thermodynamic stability. Also, if the Ala20 loop contributes to the stability of the ribonuclease A molecule, rigidification of this flexible region should stabilize the entire protein molecule. We substituted several residues in both regions without any dramatic effects on the native conformation and catalytic activity. As a result of their remarkably differing stability, the variants fell into two groups carrying the mutations: (a) A20P, S21P, A20P/S21P, S21L, or N34D; (b) L35S, L35A, F46Y, K31A/R33S, L35S/F46Y, L35A/F46Y, or K31A/R33S/F46Y. The first group showed a thermodynamic and kinetic stability similar to wild-type ribonuclease A, whereas both stabilities of the variants in the second group were greatly decreased, suggesting that the decrease in DeltaG can be mainly attributed to an increased unfolding rate. Although rigidification of the Ala20 loop by introduction of proline did not result in stabilization, disturbance of the network of hydrogen bonds and hydrophobic interactions that interlock the proposed unfolding region dramatically destabilized the ribonuclease A molecule.

Nonnatural residues can endow proteins with desirable properties. Here, replacing a proline residue that has a cis peptide bond in native ribonuclease A with 5,5-dimethyl-l-proline is shown to accelerate protein folding by 6-fold and enhance conformational stability by DeltaTm = 2.8 +/- 0.3 degrees C while having no effect on enzymatic activity. The rational use of this and other prosthetic segments could enable chemotherapeutic proteins to survive longer in vivo or retain activity after oral administration.

The virtue of the so-called 'proline concept' and the 'charge concept' for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the 'proline concept' as superior to the 'charge concept'. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

With the aim to distinguish between local and global conformational changes induced by trifluoroethanol in RNase A, spectroscopic and activity measurements in combination with proteolysis by unspecific proteases have been exploited for probing structural transitions of RNase A as a function of trifluoroethanol concentration. At >30% (v/v) trifluoroethanol (pH 8.0; 25degreesC), circular dichroism and fluorescence spectroscopy indicate a cooperative collapse of the tertiary structure of RNase A coinciding with the loss of its enzymatic activity. In contrast to the denaturation by guanidine hydrochloride, urea or temperature, the breakdown of the tertiary structure in trifluoroethanol is accompanied by an induction of secondary structure as detected by far-UV circular dichroism spectroscopy. Proteolysis with the nonspecific proteases subtilisin Carlsberg or proteinase K, both of which attack native RNase A at the Ala20-Ser21 peptide bond, yields refined information on conformational changes, particularly in the pretransition region. While trifluoroethanol at concentrations >40% results in a strong increase of the rate of proteolysis and new primary cleavage sites (Tyr76-Ser77, Met79-Ser80) were identified, the rate of proteolysis at trifluoroethanol concentrations <40% (v/v) is much smaller (up to two orders of magnitude) than that of the native RNase A. The proteolysis data point to a decreased flexibility in the surrounding of the Ala20-Ser21 peptide bond, which we attribute to subtle conformational changes of the ribonuclease A molecule. These changes, however, are too marginal to alter the overall catalytic and spectroscopic properties of ribonuclease A.

The region between the amino acids 31-46 was previously identified as being first exposed during thermal unfolding of bovine pancreatic ribonuclease A (RNase). The exchange of one amino acid (Leu35toSer) in this unfolded region of RNase is shown to have a dramatic destabilizing effect (ΔTm=9 °C). Antibodies raised against a peptide corresponding to the sequence of the labile region, S32-V43, of RNase were effective in stabilizing L35S-RNase against thermal inactivation (65 °C for 2 h) and surpassed the stabilization effect of antiRNase antibodies. An 11% contribution to the stabilizing effect of antiRNase antibodies resulted from antibodies recognizing the unfolding region of the enzyme.

The ulaG gene, located in the ula regulon, is crucial for the catabolism of l-ascorbate under anaerobic conditions and it has been proposed to encode for the putative l-ascorbate-6-P lactonase. The ulaG gene is widespread among eubacteria, including human commensal and pathogenic genera such as Escherichia, Shigella, Klebsiella and Salmonella. Here, we report the three-dimensional structures of the apoenzyme and Mn2+ holoenzyme of UlaG from E. coli to 2.6 Å resolution, determined using single-wavelength anomalous diffraction phasing and molecular replacement, respectively. The structures reveal a highly specialized metallo-β-lactamase-like fold derived from an ancient structural template that was involved in RNA maturation and DNA repair. This fold has a novel quaternary architecture consisting of a hexameric ring formed by a trimer of UlaG dimers. A mononuclear Mn2+-binding site resides at the core of the active site, which displays micromolar affinity for Mn2+ and a distorted trigonal bipyramidal coordination. The active site Mn2+ ion can be replaced by Co2+ or Zn2+, but not by Fe3+. We further show that the Mn2+ or Co2+-loaded enzyme exhibits lactonase activity towards l-ascorbate 6-P, thereby providing the first direct evidence of its catalytic role in the l-ascorbate catabolic pathway. Guided by the structural homology, we show that UlaG is able to cleave phosphodiester linkages in cyclic nucleotides, suggesting that the conservation of the fold and of the key catalytic residues allows for the evolutionary acquisition of substrate specificity for novel but related substrates.

Although highly stable toward unfolding, native ribonuclease A is known to be cleaved by unspecific proteases in the flexible loop region near Ala20. With the aim to create a protease-resistant ribonuclease A, Ala20 was substituted for Pro by site-directed mutagenesis. The resulting mutant enzyme was nearly identical to the wild-type enzyme in the near-UV and far-UV circular dichroism spectra, in its activity to 2',3'-cCMP and in its thermodynamic stability. However, the proteolytic resistance to proteinase K and subtilisin Carlsberg was extremely increased. Pseudo-first-order rate constants of proteolysis, determined by densitometric analysis of the bands of intact protein in SDS-PAGE, decreased by two orders of magnitude. In contrast, the rate constant of proteolysis with elastase was similar to that of the wild-type enzyme. These differences can be explained by the analysis of the fragments occurring in proteolysis with elastase. Ser21-Ser22 was identified as the main primary cleavage site in the degradation of the mutant enzyme by elastase. Obviously, this bond is not cleavable by proteinase K or subtilisin Carlsberg. The results demonstrate the high potential of a single mutation in protein stabilization to proteolytic degradation.

Acylformylketene S,S- and S,N-acetals 1 and 12 react with amines substituting one or both donor groups at the acetal carbon atom leading to 2-5, 7, 9-11, 13, 14 and 18 and also yielding azomethines 6-9, 11, 17 and 21. Obviously, the reaction at the acetal carbon proceeds by thermodynamic control and at the aldehyde carbon by kinetic control. 1,4-Dinucleophiles are appropriate building blocks for heterocycles 19, 20 and 22.

The push-pull systems 1 and 7 undergo regioselective cyclization with cyanothioacetamide 2 leading to pyridinethiones 3 and 8. Subsequent S-alkylation affords the alkylthio derivatives 5 and 10. Thieno[2,3-b]pyridines 6 and 11 are formed by using an alpha-CH-acidic halogen compound as alkylating agent, followed by Thorpe-Ziegler reaction. The cyano group in 6d and 11d can be converted into an imidazoline ring by treatment with 1,2-diaminoethane hydrate and carbon disulfide. Structures are supported by spectral data as well as by X-ray analysis of 5d.

Treatment of beta-keto enolates 1 with carbon disulfide in the presence of sodium hydride and subsequent alkylation yield the open-chain or cyclic acylformylketene S,S-acetals 3 and 4, respectively. Thiophenes 5, 6, or 7 and thieno[2,3-b]thiophenes 8 are formed by using as alkylating agents first methyl iodide and then an alpha-CH-acidic halo compound or only two equivalents of the latter. Beta-Keto enolates 1 also react with phenyl isothiocyanate to give acylformylketene S,N-acetals 10, aroylthiophenes 11 or thiazolidin-4-ones 12.

Treatment of o-chlorophenyl beta-ketoenolates 1 with carbon disulfide in the presence of sodium hydride and subsequent alkylation with CH-acidic halocompounds affords the thieno[2,3-b]-4H-[1]benzothiin-4-ones 3. Reaction of 1 with phenyl isothiocyanate leads to thiophenes 6 which undergo intramolecular cyclization yielding thieno[2,3-b]quinolin-4-ones 7.