Signal transducer gp130: biochemical characterization of the three membrane-proximal extracellular domains and evaluation of their oligomerization potential.

Glycoprotein 130 (gp130) is a type I transmembrane protein and serves as the common signal-transducing receptor subunit of the interleukin-6-type cytokines. Whereas the membrane-distal half of the gp130 extracellular part confers ligand binding and has been subject to intense investigation, the structural and functional features of its membrane-proximal half are poorly understood. On the basis of predictions of tertiary structure, the membrane-proximal part consists of three fibronectin-type-III-like domains D4, D5 and D6. Here we describe the bacterial expression of the polypeptides predicted to comprise each of these three domains. The recombinant proteins were refolded from solubilized inclusion bodies in vitro, purified to homogeneity and characterized by means of size-exclusion chromatography and CD spectroscopy. For the first time the prediction of three individual membrane-proximal protein domains for gp130 has been verified experimentally. The three domains do not show intermediate-affinity or high-affinity interactions between each other. Mapping of a neutralizing gp130 monoclonal antibody against D4 suggested a particular functional role of this domain for gp130 activation, because above that an intrinsic tendency for low-affinity oligomerization was demonstrated for D4.

Structure-function studies of an IGF-I analogue that can be chemically cleaved to a two-chain mini-IGF-I.

The structure and biological activities of two disulphide isomers of a C-region deletion mutant of insulin-like growth factor-I (IGF-I) which has an Asn--Gly link engineered at the junction of the A- and B-regions were studied before and after chemical cleavage. Circular dichroism (CD) spectra and binding affinity to IGF binding protein 3 (IGFBP3) indicated that the treatment with hydroxylamine did not disrupt the overall tertiary fold of the hormones. Cleavage restored some binding affinity for the IGF-I receptor in both isomers and weakly restored the ability to stimulate incorporation of tritiated thymidine into DNA in NIH 3T3 fibroblasts transfected with the human IGF-I receptor. Cleavage also restored metabolic capacity, as measured by the ability of the isomers to promote lipogenesis in isolated rat adipocytes through the insulin receptor. These results are consistent with the theory that binding of IGF-I to the IGF-I receptor requires a conformational change similar to that involved in insulin binding the insulin receptor. The weak affinity for the IGF-I receptor after cleavage is consistent with the belief that residues in the C-region interact with the IGF-I receptor. This structural difference between insulin and IGF-I gives each a higher binding affinity for its own receptor.

Structure-activity relationship of the p55 TNF receptor death domain and its lymphoproliferation mutants.

Upon stimulation with tumor necrosis factor (TNF), the TNF receptor (TNFR55) mediates a multitude of effects both in normal and in tumor cells. Clustering of the intracellular domain of the receptor, the so-called death domain (DD), is responsible for both the initiation of cell killing and the activation of gene expression. To characterize this domain further, TNFR55 DD was expressed and purified as a thioredoxin fusion protein in Escherichia coli. Circular dichroism, steady-state and time-resolved fluorescence spectroscopy were used to compare TNFR55 DD with DDs of the Fas antigen (Fas), the Fas-associating protein with DD (FADD) and p75 nerve growth factor receptor, for which the 3-dimensional structure are already known. The structural information derived from the measurements strongly suggests that TNFR55 DD adopts a similar fold in solution. This prompted a homology modeling of the TNFR DD 3-D structure using FADD as a template. In vivo studies revealed a difference between the two lymphoproliferation (lpr) mutations. Biophysical techniques were used to analyze the effect of changing Leu351 to Ala and Leu351 to Asn on the global structure and its impact on the overall stability of TNFR55 DD. The results obtained from these experiments in combination with the modeled structure offer an explanation for the in vivo observed difference.

Enhancing the T-->R transition of insulin by helix-promoting sequence modifications at the N-terminal B-chain.

Structurally, the T-->R transition of insulin mainly consists of a rearrangement of the N-terminal B-chain (residues B1-B8) from extended to helical in one or both of the trimers of the hexamer. The dependence of the transition on the nature of the ligands inducing it, such as inorganic anions or phenolic compounds, as well as of the metal ions complexing the hexamer, has been the subject of extensive investigations. This study explores the effect of helix-enhancing modifications of the N-terminal B-chain sequence where the transition actually occurs, with special emphasis on N-capping. In total 15 different analogues were prepared by semisynthesis. 80% of the hexamers of the most successful analogues with zinc were found to adopt the T3R3 state in the absence of any transforming ligands, as compared to only 4% of wild-type insulin. Transformation with SCN- ions can exceed the T3R3 state where it stops in the case of wild-type insulin. Full transformation to the R6 state can be achieved by only one-tenth the phenol concentration required for wild-type insulin, i.e. almost at the stoichiometric ratio of 6 phenols per hexamer.

The lifetime of insulin hexamers.

The kinetic stability of insulin hexamers containing two metal ions was investigated by means of hybridization experiments. Insulin was covalently labeled at the N(epsilon)-amino group of Lys(B29) by a fluorescence donor and acceptor group, respectively. The labels neither affect the tertiary structure nor interfere with self-association. Equimolar solutions of pure donor and acceptor insulin hexamers were mixed, and the hybridization was monitored by fluorescence resonance energy transfer. With the total insulin concentration remaining constant and the association/dissociation equilibria unperturbed, the subunit interchange between hexamers is an entropy-driven relaxation process that ends at statistical distribution of the labels over 16 types of hexamers differing by their composition. The analytical description of the interchange kinetics on the basis of a plausible model has yielded the first experimental values for the lifetime of the hexamers. The lifetime is reciprocal to the product of the concentration of the exchanged species and the interchange rate constant: tau = 1/(c. k). Measured for different concentrations, temperatures, metal ions, and ligand-dependent conformational states, the lifetime was found to cover a range from minutes for T(6) to days for R(6) hexamers. The approach can be used under an unlimited variety of conditions. The information it provides is of obvious relevance for the handling, storage, and pharmacokinetic properties of insulin preparations.

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.

Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1. The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.

The signal transducer gp130: solution structure of the carboxy-terminal domain of the cytokine receptor homology region.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the interleukin-6-type cytokines. It is a member of the cytokine-receptor superfamily predicted to consist of six domains in its extracellular part. The second and third domain constitute the cytokine-binding module defined by a set of four conserved cysteines and a WSXWS motif, respectively. The three-dimensional structure of the carboxy-terminal domain of this region was determined by multidimensional NMR. The domain consists of seven beta-strands constituting a fibronectin type III-like topology. The structure reveals that the WSDWS motif of gp130 is part of an extended tryptophan/arginine zipper which modulates the conformation of the CD loop.

The solution structure of a superpotent B-chain-shortened single-replacement insulin analogue.

This paper reports on an insulin analogue with 12.5-fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy. The analogue is [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. C-terminal truncation of the B-chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D-Ala for the wild-type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild-type hormone when the B-chain is present in full length. The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the "active" conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three-dimensional structure of [D-AlaB26]des-(B27-B30)-tetrapeptide-insulin-B26-amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.

Characterization and binding specificity of the monomeric STAT3-SH2 domain.

Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here we describe the expression, purification, and refolding of the STAT3-SH2 domain. Proper folding of the isolated protein was proven by circular dichroism and fluorescence spectroscopy. The STAT3-SH2 domain undergoes a conformational change upon dimerization. Using an enzyme-linked immunosorbent assay we demonstrate that the monomeric domain binds to specific phosphotyrosine peptides. The specificity of binding to phosphotyrosine peptides was assayed with the tyrosine motif encompassing Tyr705 of STAT3 and with all tyrosine motifs present in the cytoplasmic tail of the signal transducer gp130.

Analysis of protein self-association at constant concentration by fluorescence-energy transfer.

Fluorescence-resonance-energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self-association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor-labelled and acceptor-labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Forster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self-association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the epsilon-amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2-Aminobenzoyl and 3-nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Forster distance of 29 A matches their 32-A separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor-insulin and acceptor-insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K12, of 0.72x10(5) M(-1). Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.

The membrane proximal cytokine receptor domain of the human interleukin-6 receptor is sufficient for ligand binding but not for gp130 association.

Interleukin-6 (IL-6) belongs to the family of the "four-helix bundle" cytokines. The extracellular parts of their receptors consist of several Ig- and fibronectin type III-like domains. Characteristic of these receptors is a cytokine-binding module consisting of two such fibronectin domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine (WSXWS) sequence motif. On target cells, IL-6 binds to a specific IL-6 receptor (IL-6R), and the complex of IL-6.IL-6R associates with the signal transducing protein gp130. The IL-6R consists of three extracellular domains. The NH2-terminal Ig-like domain is not needed for ligand binding and signal initiation. Here we have investigated the properties and functional role of the third membrane proximal domain. The protein can be efficiently expressed in bacteria, and the refolded domain is shown to be sufficient for IL-6 binding. When complexed with IL-6, however, it fails to associate with the gp130 protein. Since the second and the third domain together with IL-6 can bind to gp130 and induce signaling, our data demonstrate the ligand binding function of the third domain and point to an important role of the second domain in complex formation with gp130 and signaling.

Interleukin-6 and related cytokines: effect on the acute phase reaction.

The acute phase response is the answer of the organism to disturbances of its physiological homeostasis. It consists of a local and a systemic reaction. The latter is characterized by dramatic changes in the concentration of some plasma proteins called acute phase proteins. Interleukin-6 (IL-6) has been identified in vitro and in vivo as the major hepatocyte stimulating factor. Subsequently, additional hepatocyte stimulating factors, such as leukemia inhibitory factor, oncostatin-M, interleukin-11 and ciliary neurotrophic factor have been discovered. IL-t and related cytokines belong to the so-called alpha-helical cytokine family characterized by four antiparallel helices. IL-6 and IL-6-type cytokines exert their action via plasma membrane receptor complexes consisting of specific cytokine binding subunits and a common signal transducing protein gp130. In this presentation we focus on structure/function studies of IL-6, its receptor subunits gp80 and gp130, the internalization of the ligand/receptor complex and a recently elucidated signal transduction pathway. We have shown that protein tyrosine kinases of the JAK family are associated with the cytoplasmic domain of gp130 and are activated in response to IL-6. Subsequently, the transcription factors--named STATs (signal transducers and activators of transcription)--STAT1 alpha and STAT3 are transiently recruited to the cytoplasmic domain of gp130, where they become tyrosine phosphorylated by JAK kinases. In addition to the tyrosine phosphorylation we have observed that IL-6 also induces a serine phosphorylation of STAT3. This modification occurs with a delayed time-course as compared to the tyrosine phosphorylation and is inhibited by the protein kinase inhibitor H7. We propose that the STAT3 serine phosphorylation is required for transactivation of IL-6 target genes which is also inhibited by H7.

Analysis of the mechanism of action of anti-human interleukin-6 and anti-human interleukin-6 receptor-neutralising monoclonal antibodies.

Anti-human interleukin-6 (human IL-6) and anti-human IL-6 receptor (IL-6R)-neutralising monoclonal antibodies (mAbs) are among the most promising human IL-6-specific inhibitors and have been shown to exert short-term beneficial effects in clinical trials. Simultaneous treatment with different anti-human IL-6 or anti-human IL-6R mAbs was recently suggested to be a potent way to inhibit the action of the cytokine in vivo. Although some of these mAbs are already used, their mechanisms of action and the location of their epitopes on the surface of human IL-6 and human IL-6R are still unknown. Here, we analysed the capacity of several anti-human IL-6 and anti-human IL-6R mAbs to inhibit the interaction between human IL-6, human IL-6R, and human glycoprotein 130 (gp130). We mapped the epitopes of several of these mAbs by studying their binding to human IL-6 and human IL-6R mutant proteins. Our results show that several anti-human IL-6 and anti-human IL-6R-neutralising mAbs block the binding between human IL-6 and human IL-6R, whereas others block the binding to gp130. We provide evidence that some of the latter mAbs inhibit interaction with gp130beta1, whereas others interfere with the binding to gp130beta2. Our results suggest that residues included in the C'D' loop of human IL-6R interact with gp130beta2.

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure.

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

Exclusion of bioactive contaminations in Streptococcus pyogenes erythrogenic toxin A preparations by recombinant expression in Escherichia coli.

The streptococcal erythrogenic exotoxin A (SPEA) belongs to the family of bacterial superantigens and has been implicated in the pathogenesis of a toxic shock-like syndrome and scarlet fever. Concerning its biological activity, mainly T-cell-stimulatory properties, conflicting data exist. In this study, we show that most of the SPEA preparations used so far contain biologically active contaminations. Natural SPEA from the culture supernatant of Streptococcus pyogenes NY-5 and recombinant SPEA purified from the culture filtrate of S. sanguis are strongly contaminated with DNases. We show that natural SPEA induces more tumor necrosis factor alpha (TNF-alpha) than recombinant SPEA, but we also show that DNases are able to induce TNF-alpha. In commercial SPEA preparations, we identified a highly active protease, which was shown not to be SPEB. To exclude these contaminations, we overexpressed SPEA cloned in the effective high-level expression vector pIN-III-ompA2 in Escherichia coli. The expressed SPEA shows the same amino acid composition as natural SPEA, whereas functional studies reported so far were carried out with toxins containing an incorrect amino terminus. We describe the rapid purification of lipopolysaccharide-, DNase-, and protease-free SPEA in two steps from the host's periplasm and its structural characterization by circular dichroism. Our results represent for the first time the production in E. coli of recombinant SPEA with the authentic N-terminal sequence and a proven superantigenic activity. Collectively, our results indicate that immunological studies of superantigens require highly purified substances free of biologically active contaminations.

Molecular modeling-guided mutagenesis of the extracellular part of gp130 leads to the identification of contact sites in the interleukin-6 (IL-6).IL-6 receptor.gp130 complex.

The transmembrane protein gp130 is involved in many cytokine-mediated cellular responses and acts therein as the signal-transducing subunit. In the case of interleukin-6 (IL-6), the signal-transducing complex is composed of the ligand IL-6, the IL-6 receptor (IL-6R, gp80, CD126), and at least two gp130 (CD130) molecules. The extracellular part of the signal transducer gp130 consists of six fibronectin type III-like domains. It has recently been shown that the three membrane distal domains bind to the IL-6. IL-6R complex. A structural model of the IL-6.IL-6R.gp130 complex enabled us to propose amino acid residues in these domains of gp130 interacting with IL-6 bound to its receptor. The proposed amino acid residues located in the B'C' loop (Val252) and in the F'G' loop (Gly306, Lys307) of domain 3 and in the hinge region (Tyr218) connecting domains 2 and 3 of gp130 were mutated to disturb ternary complex formation. Binding of wild type and mutants of the extracellular region of gp130 was studied by use of a co-precipitation assay and Scatchard analysis. All mutants showed decreased binding to the IL-6.IL-6R complex. Biological function of the membrane-bound gp130 mutants was studied by STAT (signal transducer and activator of transcription) activation in COS-7 cells and by proliferation of stably transfected Ba/F3 cells. Reduced binding of the mutants was accompanied by decreased biological activity. The combined approach of molecular modeling and site-directed mutagenesis has led to the identification of amino acid residues in gp130 required for complex formation with IL-6 and its receptor.

The signal transducer gp130--bacterial expression, refolding and properties of the carboxy-terminal domain of the cytokine-binding module.

Gp130 is the signal transducing receptor subunit of the so-called interleukin-6-type cytokines. This transmembrane protein is a member of the cytokine-receptor superfamily predicted to consist of six fibronectin-type-III-like domains in its extracellular part. The second and the third domain constitute the so-called cytokine-binding module. Domain 2 is characterized by a set of four conserved Cys residues, domain 3 by a conserved WSXWS motif. As a first approach to a more detailed characterization of the cytokine-binding domains of human gp130, we have expressed in Escherichia coli two forms of domain 3 differing in length. Both proteins were purified and refolded in a single step applying size-exclusion chromatography. According to the rotational correlation times deduced from fluorescence anisotropy decay, they do not form aggregates. CD and fluorescence spectroscopy were used to study thermal unfolding and denaturation by guanidinium hydrochloride. It was shown that N- and C-terminal extension by residues of the adjacent hinge regions substantially increase the thermal stability of the domain, which is conceivable from a molecular model. These results are the basis for further structural investigation by NMR spectroscopy.

Recombinant human O6-alkylguanine-DNA alkyltransferase induces conformational change in bound DNA.

Circular dichroism, and steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT bound to ds-DNA. Contrary to fluorescence, analysis of the far-UV CD spectra indicated a conformational change of AGT upon binding to DNA: its alpha-helical content is increased by approximately 12%. Analysis of near-UV CD spectra revealed that DNA was also affected, probably being separated into single strands locally.

Recombinant human O6-alkylguanine-DNA alkyltransferase (AGT), Cys145-alkylated AGT and Cys145 --> Met145 mutant AGT: comparison by isoelectric focusing, CD and time-resolved fluorescence spectroscopy.

Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.

Calculations of the CD spectrum of bovine pancreatic ribonuclease.

CD spectra of bovine pancreatic ribonuclease A (RNase A) and its subtilisin-modified from (RNase S) have been calculated, based upon high-resolution structures from x-ray diffraction. All known transitions in the peptide and side-chain groups, especially the aromatic and disulfide groups, have been included. Calculations have been performed with both the matrix method and with first-order perturbation theory. A newly developed method for treating the electrostatic interactions among transition charge densities and between static charge distributions and transition charge densities is used. The effects of local electrostatic fields upon the group transition energies are included for all transitions. Rotational strengths generated by the matrix method were combined with Gaussian band shapes to generate theoretical CD spectra. The calculated spectra reproduce the signs and approximate magnitudes of the near-uv CD bands of both RNase A and S. Agreement is most satisfactory for the negative 275 nm band, dominated by tyrosine contributions. In agreement with two previous studies by other workers, coupling between Tyr 73 and Tyr 115 is the single most important factor in this band. The positive band observed near 240 nm is dominated by disulfide contributions, according to our results. The far-uv CD spectrum is poorly reproduced by the calculations. The observed 208 nm band, characteristic of alpha-helices, is absent from the calculated spectrum, probably because the helices in RNase are short. A strong positive couplet centered near 190 nm is predicted but not observed. Possible reasons for these incorrect predictions of the current theoretical model in the far-uv are discussed.