Hirudin is the most potent and specific inhibitor of thrombin, a key enzyme in the coagulation process existing in equilibrium between its procoagulant (fast) and anticoagulant (slow) form. In a previous study, we described the solid-phase synthesis of a Trp3 analogue of fragment 1-47 of hirudin HM2, which displayed approximately 5-fold higher thrombin inhibitory potency relative to that of the natural product [De Filippis, V., et al. (1995) Biochemistry 34, 9552-9564]. By combining automated and manual peptide synthesis, here we have produced in high yields seven analogues of fragment 1-47 containing natural and non-natural amino acids. In particular, we have replaced Val1 with tert-butylglycine (tBug), Ser2 with Arg, and Tyr3 with Phe, cyclohexylalanine (Cha), Trp, alpha-naphthylalanine (alphaNal), and beta-naphthylalanine (betaNal). The crude reduced peptides are able to fold almost quantitatively into the disulfide-cross-linked species, whose unique alignment (Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37) has been shown to be identical to that of the natural fragment. The results of conformational characterization provide evidence that synthetic peptides retain the structural features of the natural species, whereas thrombin inhibition data indicate that the synthetic analogues are all more potent inhibitors of thrombin. In particular, Val --> tBug exchange leads to a 3-fold increase in binding, interpreted as arising from a favorable reduction of the entropy of binding, due to the presence of the more symmetric side chain of tBug relative to that of Val. The S2R analogue binds 24- and 125-fold more tightly than the natural fragment to the fast or slow form of thrombin. These results are explained by considering that Arg2 may favorably couple to Glu192, a key residue involved in the slow to fast transition, thus stabilizing the slow form. Replacement of Tyr3 with more hydrophobic residues having different side chain orientations and electronic structures improves binding by 2-40-fold, suggesting that nonpolar interactions and shape-dependent packing effects strongly influence binding at this position. Overall, these results provide new insights for elucidating the mechanism of hirudin-thrombin recognition at the molecular level and highlight new strategies for designing more potent and selective inhibitors of thrombin.
Cyclic mono-cystinyl active-site fragments of thioredoxin and thioredoxin reductase were synthesized as N-acetyl and C-amide octapeptides by conventional methods of peptide synthesis in solution and on solid supports. Using a side-chain protection based on acid-labile tert-butanol-derived groups and on the S-tert-butylthio unsymmetric disulfide for the thiol functions, in combination with N alpha-Z- or N alpha-Nps derivatives in the chain elongation steps, the synthesis in solution was carried out in straightforward manner yielding the fully protected octapeptides as well characterized compounds. Upon deprotection with trifluoroacetic acid and reduction of the unsymmetrical disulfides with tri-butylphosphine, the resulting bis-cysteinyl-octapeptides were oxidized in dimethylformamide with azodicarboxylic acid di-tert-butyl ester to produce the desired cyclic compounds in good overall yields. For the synthesis on solid supports a similar acid-labile side-chain protection was applied in combination with the N alpha 9-flourenylmethyoxycarbonyl derivatives in the chain elongation steps. Thereby acylations were performed with the related amino acid N-carboxyanhydrides (UNCAs) or by the O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetraf luoroborate/1- hydroxybenzotriazole (TBTU/HOBt) procedure. The solid phase synthesis of the two octapeptides led to unexpected difficulties in terms of recovery of peptidic material from the resins in the final acidolytic cleavage step as well as of racemization at the level of the cysteine residues by the TBTU/HOBt coupling method. Racemization was efficiently suppressed by employing the related pentafluorophenyl ester and this method led to crude octapeptide products of a degree of purity comparable to those obtained by the synthesis in solution. However, the recovery of the peptides from the resin, i.e., irreversible reattachment of cleaved peptidic material via alkylation of various side-chain functions, could not be avoided even using the most efficient scavengers or their cocktails.
Enkephalin represents one of the bioactive peptide molecules most extensively investigated both in solution and in the crystal state. Depending upon the environment chosen for such studies, three main conformational states were identified for this flexible, linear pentapeptide—i.e., an extended conformation, a single‐bend, and a double‐bend structure. Since CD and Fourier transform ir (FTIR) spectra of Leu‐enkephalin solubilized in negatively charged reverse micelles of bis (2‐ethylhexyl)sulfosuccinate sodium salt/isooctane/water were supportive of a restricted conformational space of the aromatic side chains and of a bended type fold, we have analyzed by nmr the conformational preferences of Leu‐enkephalin in reverse micelles using a synthetic [13C, 15N]‐backbone‐labeled sample. The overall conformation derived from nuclear Overhauser effect spectroscopy (NOESY) and 15N‐filtered rotating frame NOESY (ROESY) spectra and by distance geometry calculations is a double‐bend fold of the backbone that is comparable to one of the known x‐ray structures. Thereby the tyrosine side chain is inserted into the hydrophobic core of the reverse micelles in a restrained conformational space as well evidenced by NOEs between the aromatic ring protons and the surfactant. The proximity of the aromatic rings of tyrosine and phenylalanine indicate a preferred structure consistent with the postulated conformation of the opioid peptide in the δ‐receptor‐bound state. These results confirm the interesting and promising properties of reverse micelles as membrane mimetica. © 1997 John Wiley & Sons, Inc. Biopoly 41: 591–606, 1997
The conformation of basic fatty acid binding protein from chicken liver and the binding properties of the apo protein toward 11-dansylamino-undecanoic acid were investigated by CD and fluorescence spectroscopy. In one set of experiments the binding process was followed by the appearance of induced optical activity in the absorption region of the dansyl chromophore. In a second set of experiments the binding process was followed by the large enhancement of emission fluorescence of the dansyl fluorophore. From the saturation curves, the stoichiometry of the complex and the binding constant of the fatty acid to the protein were precisely determined. The values of the dissociation constant determined with the two methods were in excellent agreement: we obtained KD = (1.0 +/- 0.1) x 10(-6) M in a 0.9: 1 stoichiometry. The native conformation of the protein is remarkably stable in a variety of solvent systems, including acetonitrile-water, ethylene glycol-water, and dioxane-water of various compositions. The CD results also showed that the binding of the fatty acid does not induce any appreciable change in the protein conformation. In a mixture of water and 2,2,2-trifluoroethanol 1:9 (v/v), the native conformation collapses and a new ordered structure is formed, characterized by a high amount of alpha-helix.
Bis(cysteinyl)octapeptides related to the active sites of the oxidoreductases protein disulfide isomerase (PDI), thioredoxin reductase (trr), glutaredoxin (grx), and thioredoxin (trx) were analyzed for their propensity to form the intramolecular 14-membered disulfide ring in oxidation experiments. The rank order of percentage of cyclic monomer formed in aqueous buffer (pH 7.0) at 10(-3) M concentration was found to be very similar, but opposite to that of the Kox and, correspondingly, of the redox potentials of the native enzymes. Attempts to induce intrinsic conformational preferences of the peptides by addition of trifluoroethanol led to enhancements of beta-turn structures as reflected by the CD and Fourier transform ir spectra. The induced secondary structure, instead of aligning the tendencies of the excised fragments for loop formation with those of the intact proteins, was found to suppress the differences by significantly increasing the preference for cyclic monomers (approximately 90%). Similarly, operating under denaturing conditions, i.e., in 6 M guanidinium hydrochloride, only for the trx peptide was the statistical product distribution obtained. For the remaining peptides, again a strong increase of cyclic monomer contents was observed that could not be correlated with dissolution of beta-sheet type aggregates. The CD spectra are more consistent with the presence of ordered structure to some extent, possibly resulting from an hydrophobic collapse of the sparingly soluble peptides. The results of the oxidation experiments further support previous findings from thiol disulfide interchange equilibria, which clearly revealed a decisive role of the characteristic thioredoxin structural motif in dictating the redox properties of the enzymes. Point mutations in the active sites of the oxidoreductases allowed us to affect their redox potentials strongly, but apparently only in the constraint form of the three-dimensional structure as similar exchanges in the excised fragments did not produce the expected effect. This observation contrasts with numerous reports that the conformation of short disulfide loops is mainly dictated by the amino acid sequence.
Type-I 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 Ij-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 PAl-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 Ij-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.Keywords; serpin ; circular dichroism ; Fourier-transform infrared spectroscopy ; serine proteinase ; structure.The 54 000-kDa type-I plasminogen-activator inhibitor (PAI-1) is the primary inhibitor of the two types of plasminogen activators, urokinase-type (uPA) and tissue-type. As such, it is an important modulator of extracellular proteolysis, turnover of the extracellular matrix and fibrinolysis (Andreasen et al., 1990). PAI-1 belongs to the serpin (serine-proteinase inhibitor) superfamily. An exclusive feature of PAT-1 is its transition to a latent form. This functionally inactive form of PAI-1 is formed when PAI-1 is purified in the absence of its protein cofactor vitronectin (Preissner and Jenne, 1991). Activation of latent PAI-1 can be achieved by denaturation with SDS or chaotropic agents such as guanidine hydrochloride and refolding after removal of the denaturing agent (Hekman and Loskutoff, 1985).The inhibition reaction for serpins in general is characterized by formation of a stable complex with 1 : 1 stoichiometry. The active site of the proteinase reacts with the reactive-center peptide bond, localized in an approximately 20-amino-acid-long peptide stretch termed the reactive-center loop (RCL), which is exposed on the surface of the serpins. Under some conditions, serine proteinase . serpin complexes dissociate to active proteinases and inactive serpins that are proteolytically cleaved in the reactive-center peptide bond. Conformational changes after reactive-center cleavage have been detected by measurement of thermal stability and spectroscopic methods such as CD and fluorescence emission with many serpins (Huber and Carrell, 1989...
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