Determinants of substrate recognition by poliovirus 2A proteinase.

Poliovirus proteinase 2A (2Apro) is autocatalytically released from the viral polyprotein by cleavage in cis of a Tyr-Gly dipeptide at its own amino terminus, resulting in separation of the P1 structural and P2-P3 nonstructural protein precursors. A second Ty-Gly dipeptide within 3D polymerase is cleaved by 2Apro in trans, but this is not essential for viral proliferation. The mechanism which limits cleavage to only 2 of the 10 Tyr-Gly dipeptides within the poliovirus polyprotein has not been characterized. We have therefore undertaken a systematic mutational analysis of the VP1-2A site to elucidate determinants of substrate recognition by 2Apro. The P2 and P1' positions are important determinants for cis cleavage of this site, whereas a variety of substituents could be tolerated at the P2', P1, and P3 positions. The requirements for trans cleavage of this site were more stringent. We found that the 2Apro of coxsackievirus type A21 and rhinoviruses 2 and 14 have stringent requirements similar to those of poliovirus 2Apro for cleavage in trans.     

Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.     

Thermodynamic origin of cis/trans isomers of a proline-containing beta-turn model dipeptide in aqueous solution: a combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear Overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-turn model dipeptide [13C]-Ac-L-Pro-D-Ala-NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H-nmr, two-dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one-dimensional steady-state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random-coil peptides in H2O (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-AlaNH and AlaNH-NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C-CONH(CH3) fragment exhibits significant conformational flexibility; however, a low-energy conformer resembles closely the beta II-turn folded conformations of the x-ray structure of the related model peptide trans-BuCO-L-Pro-Me-D-Ala-NHMe. On the contrary, the cis isomer adopts open conformations. Steady-state intermolecular solute-solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H-nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -5.47 J.K-1.mole-1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence-specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Selective production of cis-9,trans-11 isomer of conjugated linoleic acid from trans-vaccenic acid methyl ester by Delacroixia coronata

Aims:  Bio-process development for isomer selective and efficient production of cis -9, trans -11-octadecadienoic acid (CLA) from trans -vaccenic acid ( t -VA, trans -11-octadecenoic acid) through microbial fatty acid Δ9-desaturation reaction.
Methods and Results:  A total of 550 strains of fungi and yeasts were screened for CLA production from t -VA through Δ9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t -VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t -VA ( t -VAME). Under the optimal conditions with 33·3 mg ml⁻¹ of t -VAME as substrate, 10·5 mg ml⁻¹ CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5·0%), tryptone (2·0%) and thiourea (0·83 μmol ml⁻¹). The strain produced the cis -9, trans -11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans -9, trans -11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA).
Conclusions:  A practical bio-process for selective production of cis -9, trans -11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established.
Significance and Impact of the Study:  Isomer selective bio-process for the practical production of cis -9, trans -11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.     

Photocyclization of trans-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene: evidence for adiabatic 1trans*-->1cis* photoisomerization.

The photochemistry of trans- and cis-1-(1'-naphthyl)-2-(3-hydroxyphenyl)ethene in cyclohexane and acetonitrile was examined. In cyclohexane fluorescence is the main deactivation channel for the 1trans* isomer while photocyclization is the main reaction of the 1cis* isomer. The weighty formation of hydroxychrysene following one photon absorption by the trans isomer furnished evidence of an adiabatic 1trans*-->1cis* isomerization. The photoreactivity data in acetonitrile indicated the influence of solvent polarity on the shape of the excited state surface.     

A conserved cis peptide bond is necessary for the activity of Bowman-Birk inhibitor protein

The Bowman-Birk inhibitor (BBI) family of protease inhibitors has an inhibitory region comprising a disulfide-linked nine-residue loop that adopts the characteristic canonical motif found in many serine protease inhibitors. A unique feature of the BBI loop is the presence of a cis peptide bond at the edge of the inhibitory loop. BBI-related protein fragments that encapsulate this loop retain the structure and inhibitory activity of the parent protein. The most common BBI loop sequence has a proline-proline element with a cis-trans geometry at P3'-P4'. We have examined this element by analysis of the inhibitory activity and structure for a series of synthetic fragments where each of these proline residues has been systematically replaced with alanine. The results show that only when a proline is present at P3' are potent inhibition and a cis peptide bond at that position in the solution structure observed, suggesting that this conformation is required for biological activity. Though a P4' proline is not essential for activity, it effectively stabilizes the cis conformation at P3' by suppressing alternative conformations. This is most evident from the Pro-Ala variant, which comprises a 1:1 mixture of slowly exchanging and structurally different cis and trans isomers. Monitoring the action of trypsin on this mixture by NMR shows that this protease interacts selectively with the cis P3' structure, providing direct evidence for the link between activity and the nativelike structure of the cis isomer. This is, to the best of our knowledge, the first example where cis isomer selectivity can be demonstrated for a proteinase.     

The trans-to-cis proline isomerization in E. coli Trx folding is accelerated by trans prolines

The conserved fold of thioredoxin (Trx)-like thiol/disulfide oxidoreductases contains an invariant cis-proline residue (P76 in Escherichia coli Trx) that is essential for Trx function and that is responsible for the folding rate-limiting step. E. coli Trx contains four additional prolines, which are all in the trans conformation in the native state. Notably, a recent study revealed that replacement of all four trans prolines in Trx by alanines (Trx variant Trx1P) further slowed the rate-limiting step 25-fold, indicating that one or several of the four trans prolines accelerate the trans-to-cis transition of P76 in Trx wild-type (wt). Here, we characterized the folding kinetics of Trx variants containing cisP76 and one or several of the natural trans prolines of Trx wt with NMR spectroscopy. First, we demonstrate that the isomerization reaction in Trx1P is a pure two-state transition between two distinct tertiary structures, in which all observed NMR resonances changes follow the same first-order kinetics. Moreover, we show that trans-P68 is the critical residue responsible for the faster folding of wt Trx relative to the single-proline (P76) variant Trx1P, as the two-proline variant Trx2P(P76P68) already folds seven times faster than Trx1P. trans-P34 also accelerates trans-to-cis isomerization of P76, albeit to a smaller extent. Overall, the results demonstrate that trans prolines can significantly modulate the kinetics of rate-limiting trans-to-cis proline isomerization in protein folding. Finally, we discuss possible mechanisms of acceleration and the potential significance of a protein-internal folding acceleration mechanism for Trx in a living cell.     

A cis-prolyl peptide bond isomerization dominates the folding of the alpha subunit of Trp synthase,a TIM barrel protein

The cis/trans isomerization of prolyl peptide bonds has been suggested to dominate the folding of the alpha subunit of tryptophan synthase from Escherichia coli (alphaTS). To test the role of the unique cis isomer between Asp27 and Pro28, the folding properties of P28A, P28G and G(3)P28G, a three-glycine insertion mutant between Asp27 and Gly28, were investigated using urea as a denaturant. Circular dichroism analysis demonstrated that none of the mutations perturb the secondary structure significantly, although the aromatic side-chain packing is altered for P28A and P28G. All three mutant proteins inherited the three-state thermodynamic behavior observed in wild-type alphaTS, ensuring that the fundamental features of the energy surface are intact. Kinetic studies showed that neither alanine nor glycine substitutions at Pro28 results in the elimination of any slow-refolding phases. By contrast, the G(3)P28G mutant eliminates the fastest of the slow-refolding phases and one of the two unfolding phases. Double-jump experiments on G(3)P28G confirm the assignment of the missing refolding phase to the isomerization of the Asp27-Pro28 peptide bond. These results imply that the local stability conveyed by the tight, overlapping turns containing the cis peptide bond is sufficient to favor the cis isomer for several non-prolyl residues. The free energy required to drive the isomerization reaction is provided by the formation of the stable intermediate, demonstrating that the acquisition of structure and stability is required to induce subsequent rate-limiting steps in the folding of alphaTS.     

Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease.

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.     

Structure-dependent in vitro cytotoxicity of the isomeric complexes [Ru(L)2Cl2] (L=o-tolylazopyridine and 4-methyl-2-phenylazopyridine) in comparison to [Ru(azpy)2Cl2]

The dichlorobis(2-phenylazopyridine)ruthenium(II) complexes, [Ru(azpy)(2)Cl(2)], are under renewed investigation due to their potential anticancer activity. The three most common isomers alpha-, beta- and gamma-[RuL(2)Cl(2)] with L= o-tolylazopyridine (tazpy) and 4-methyl-2-phenylazopyridine (mazpy) (alpha indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual cis, trans, cis positions, beta indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual cis, cis, cis positions, and gamma indicating the coordinating Cl, N(pyridine) and Nazo atoms in mutual trans, cis, cis positions) are synthesized and characterized by NMR spectroscopy. The molecular structures of gamma-[Ru(tazpy)(2)Cl(2)] and alpha-[Ru(mazpy)(2)Cl(2)] are determined by X-ray diffraction analysis. The IC(50) values of the geometrically isomeric [Ru(tazpy)(2)Cl(2)] and [Ru(mazpy)(2)Cl(2)] complexes compared with those of the parent [Ru(azpy)(2)Cl(2)] complexes are determined in a series of human tumour cell lines (MCF-7, EVSA-T, WIDR, IGROV, M19, A498 and H266). These data unambiguously show for all complexes the following trend: the alpha isomer shows a very high cytotoxicity, whereas the beta isomer is a factor 10 less cytotoxic. The gamma isomers of [Ru(tazpy)(2)Cl(2)] and [Ru(mazpy)(2)Cl(2)] display a very high cytotoxicity comparable to that of the gamma isomer of the parent compound [Ru(azpy)(2)Cl(2)] and to that of the alpha isomer. These biological data are of the utmost importance for a better understanding of the structure-activity relationships for the isomeric [RuL(2)Cl(2)] complexes.     

A comparative study of the physical properties and enzymatic reactions of slow reacting substance of anaphylaxis and some leukotriene D4 isomers

Eight synthetic isomers of hydroxy-6-S-cysteinylglycine -7,9,11,14-eicosatetraenoic acid were compared with authentic guinea pig SRS-A using UV spectroscopy, high performance liquid chromatography and soybean lipoxygenase. It was found that only the 5S, 6R 7, 9trans 11,14cis isomer was similar to SRS-A in all respects. The 5S, 6R 7trans, 9,11,14 cis isomer shows similar UV and HPLC characteristics but differs in that it spontaneously undergoes a 1,7 hydride shift reaction and unexpectedly does not react with soybean lipoxygenase.     

NMR investigations of proline and its derivatives. 4-Proton magnetic resonance parameters and structure of acetyl-proline amide.

The proton magnetic resonance (PMR) spectrum of acetyl-proline amide in D2O solution has been analysed by computer simulation. The spectra of the cis and the trans isomers have been separated and their PMR parameters (chemical shift and coupling constants) are given. Vicinal coupling constants of the pyrrolidine ring are interpreted by means of a Karplus zone relation. The chemical shift effect of the anisotropy of both peptide planes is considered. It follows that both isomers are puckered with Cgamma in an endo position, but the cis isomer is more rigid than the trans isomer, which moreover undergoes a small interconversion of the Cgamma and Cdelta atoms between two extreme spatial positions. The dihedral angle phi has different values in both isomers. Thus, the dihedral angle between the two peptide planes is smaller in the trans isomer than in the cis isomer.     

Energetics of assembling an artificial heterodimer with an alpha/beta motif: cleaved versus uncleaved Escherichia coli thioredoxin.

We have studied the folding/binding process between the N- and C-fragments (1-73, 74-108) of oxidized Escherichia coli thioredoxin (Trx) to compare the energetics between the cleaved and uncleaved Trx. Sedimentation equilibrium analysis in 0.1 M potassium phosphate, pH 5.7, shows (i) the strong and weak self-association of the N- and C-fragments, respectively, (ii) a heterodimer with a small dissociation constant (K(d)) ca. 100 nM, and (iii) monomeric Trx. To avoid self-association, measurements were carried out in 10 mM potassium phosphate, pH 5.7. Far-UV CD spectra of the fragments at variable temperature show an isodichroic point at 208 nm and a non-cooperative cold induced disordering transition without concentration dependence. Deconvolution of these spectra indicates the presence of residual structure. Titration of the N-fragment with an excess of C-fragment indicates a 1:1 stoichiometric complex with an apparent K(d) ca. 49 nM. Analysis of this complex by CD and hydrogen exchange/2D-NMR (Tasayco and Chao (1995) Proteins: Struct., Funct., Genet. 22, 41-44) spectroscopy indicates the reassembly of the alpha/beta motif of Trx. GnHCl induced unfolding measurements give DeltaG(0) values of 9.5 +/- 0.2 and 10.0 +/- 0.4 kcal/mol at 20 degrees C for the uncleaved and cleaved Trx, respectively. The far-UV CD melting curve of uncleaved Trx indicates an intriguing non-cooperative upward baseline trend. CCA analysis of these spectra indicates the presence of a native-like folded intermediate. A three-state thermodynamic analysis of the thermal transition curves gives a total DeltaH(0) of unfolding of 121 +/- 4 kcal/mol at the T(m) (88 degrees C), while the two-state analysis for cleaved Trx gives 122 +/- 6 kcal/mol at 88 degrees C. Analysis of the chemical and thermal unfolding of both proteins indicates a value of ca. 1 M for the apparent effective concentration (C(eff)) of cleaved Trx.     

Evidence that the substrate backbone conformation is critical to phosphorylation by p42 MAP kinase

The effect of prolyl bond isomers on the substrate recognition capabilities of various endoproteases may be investigated in a reaction where both cis/trans isomers co-exist. Here we address the question of whether enzyme reactions at the side chain of an amino acid preceding proline proceed through an isomer specific pathway. The proline-directed p42 mitogen-activated protein kinase (ERK2) was used to phosphorylate the serine side chain in Pro-Arg-Ser-Pro-Phe-4-nitroanilide under conditions where different amounts of cis prolyl isomer of the substrate were present. Initial phosphorylation rates were calculated ranging between zero at 100% cis isomer and around 60 pM/min at the equilibrium content of 83.5% trans isomer. In the presence of the peptidyl-prolyl cis/trans isomerase human hFKBP12 (500 nM), cis/trans isomerization proceeds rapidly, permitting the maximal phosphorylation rate to be observed in the dead time of the experiment. Results show that correct signature sequences are not sufficient to render potential substrates reactive to proline-directed enzymatic phosphorylations, but that the conformational state of the peptide bond following serine (threonine) is a critical determinant. Therefore, catalysis by peptidyl-prolyl cis/trans isomerases may add a new level of control to intracellular protein phosphorylations.     

Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn

A systematic examination by 1H nuclear magnetic resonance of the population of beta-turn-containing conformers in several series of short linear peptides in water solution has demonstrated a dependence on amino acid sequence which has important implications for initiation of protein folding. The peptides consist of a number of variants of the sequence Tyr-Pro-Tyr-Asp, the trans isomer of which was previously shown to contain a reverse turn in water. Two-dimensional rotating-frame nuclear Overhauser effect spectroscopy provides unequivocal evidence that substantial populations of reverse turn conformations occur in water solutions of certain of these peptides. In the unfolded state, the peptides adopt predominantly extended chain (beta) conformations in water. It appears probable from the nuclear Overhauser effect connectivities observed that the reverse turns in the trans isomers are predominantly type II. The low temperature coefficient of the amide proton resonance of the residue at position 4 of the turn suggests the presence of an intramolecular hydrogen bond. The presence of the beta-turn conformation has been confirmed for certain peptides by circular dichroism measurements. Substitutions at positions 3 and 4 in the sequence Tyr-Pro-Tyr-Asp-Val can enhance or abolish the beta-turn population in the trans peptide isomers. The residue at position 3 of the turn is the primary determinant of its stability. A small amount of additional stabilization appears to result from an electrostatic interaction between the side-chain of residue 4 and the unblocked amino terminus. For peptides of the series Tyr-Pro-X-Asp-Val, where X represents all L-amino acid except Trp and Pro, the temperature coefficient of the Asp4 amide proton resonance provides a measure of the beta-turn population. The beta-turn populations in water solution measured in this way correlate with the beta-turn probabilities determined from protein crystal structures. This indicates that it is frequently the local amino acid sequence, rather than medium- to long-range interactions in the folded protein, that determines the beta-turn conformation in the folded state. Such sequences are excellent candidates for protein folding initiation sites. A high population of structured forms appears to be present in the cis isomer of certain of the peptides, as shown by a considerable increase in the proportion of the cis isomer and by measurement of nuclear Overhauser effects and 3JN alpha coupling constants.(ABSTRACT TRUNCATED AT 400 WORDS)     

Unusual thionation of a cyclic hexapeptide. Conformational changes and dynamics.

One carbonyl oxygen of the cyclic hexapeptide cyclo(-Gly1-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=] of Gly1 but that of Phe5 which was substituted and cyclo(-Gly1-Pro2-Phe3-Val4-Phe5 psi [CS-NH]Phe6-) (B) was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe5 and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a cis Gly1-Pro2 bond in a beta VIa-turn (minor) and a trans Gly-Pro bond in a beta II'-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOEs, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a beta I- and beta II-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1 rho) were measured to verify the assumption of this fast beta I/beta II equilibrium within each isomer. Significant contributions to R1 rho-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.     

Specificity of the hepatitis C virus NS3 serine protease: effects of substitutions at the 3/4A, 4A/4B, 4B/5A, and 5A/5B cleavage sites on polyprotein processing.

Cleavage at four sites (3/4A, 4A/4B, 4B/5A, and 5A/5B) in the hepatitis C virus polyprotein requires a viral serine protease activity residing in the N-terminal one-third of the NS3 protein. Sequence comparison of the residues flanking these cleavage sites reveals conserved features including an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position. In this study, we used site-directed mutagenesis to assess the importance of these and other residues for NS3 protease-dependent cleavages. Substitutions at the P7 to P2' positions of the 4A/4B site had varied effects on cleavage efficiency. Only Arg at the P1 position or Pro at P1' substantially blocked processing at this site. Leu was tolerated at the P1 position, whereas five other substitutions allowed various degrees of cleavage. Substitutions with positively charged or other hydrophilic residues at the P7, P3, P2, and P2' positions did not reduce cleavage efficiency. Five substitutions examined at the P6 position allowed complete cleavage, demonstrating that an acidic residue at this position is not essential. Parallel results were obtained with substrates containing an active NS3 protease domain in cis or when the protease domain was supplied in trans. Selected substitutions blocking or inhibiting cleavage at the 4A/4B site were also examined at the 3/4A, 4B/5A, and 5A/5B sites. For a given substitution, a site-dependent gradient in the degree of inhibition was observed, with a 3/4A site being least sensitive to mutagenesis, followed by the 4A/4B, 4B/5A, and 5A/5B sites. In most cases, mutations abolishing cleavage at one site did not affect processing at the other serine protease-dependent sites. However, mutations at the 3/4A site which inhibited cleavage also interfered with processing at the 4B/5A site. Finally, during the course of these studies an additional NS3 protease-dependent cleavage site has been identified in the NS4B region.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.