Metal ion-induced conformational changes of phosphorylated fragments of human neurofilament (NF-M) protein.

The NF-M subunit of human neurofilaments has a C-terminal repeating 13-mer sequence. The 13-mer (Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly) (NF-M13) and 17-mer (Glu-Glu-Lys-Gly)-(NF-M13) sequences were synthesized, as were both the mono- and diphosphorylated Ser species. Circular dichroism (c.d.) studies and c.d. titrations with Al3+ and Ca2+ were performed. The conformation of the phosphorylated and unphosphorylated material was random in water. Deconvolution of the c.d. spectra, in trifluoroethanol, of the untitrated samples yielded a high content of unordered structure, similar to the poly-L-proline II structure. Titration of the phosphorylated species with Al3+ or Ca2+ caused a surprising conformational change to occur, yielding a high content of beta-pleated sheet structure. A mechanism of metal binding to the phosphofragments is proposed which may be relevant to the formation of neurofibrillary tangles in Alzheimer's disease.

Modulation of peptide specific T cell responses by non-native flanking regions.

The deduced core (75RYPNVTI81) from a T-cell stimulatory epitope of the 38 kDa protein of M. tuberculosis was studied to identify the structural elements required for the creation of a synthetic peptide antigen from an epitope core, which alone was not capable of inducing CD4+ T-cell responses. Peptides were prepared with extensions composed of native and/or non-native sequences to clarify the role of the flanking regions adjacent to the epitope core. Their binding to isolated H-2-Ab MHC glycoprotein as well as T-cell stimulatory capacity were assayed using a specific murine hybridoma T-cell line [38.H6], lymph node cells from the native 20-mer peptide primed C57BL/10 mice and human PBMCs from sensitised individuals. Elongation of the epitope core by four alanines at both N- and C-terminals resulted in a 15-mer peptide A4-75-81-A4 which was stimulatory for hybridoma T-cells and showed a small decrease in H-2-Ab binding. Substitution of one Ala by Ser in the N-terminal flank had pronounced effect and peptide A2SA-75-81-A4 proved to be more effective than the native 20-mer sequence in the hybridoma as well as in the LN cell proliferation assays. The binding of this peptide and that of the native one were similar. Testing in human PBMC cultures from eight PPD positive individuals showed that in 50% of the donors' cells responded to the 'artificial' A2SA-75-81-A4 peptide. These results suggest that it is possible to construct simple, synthetic CD4+ T-cell stimulatory peptides of high potency from a non-stimulatory, 'silent' epitope core by addition of flanking residues not part of the native sequence.

Inverse stereoselectivity in the binding of acenocoumarol to human serum albumin and to alpha 1-acid glycoprotein.

Stereoselective binding of rac-acenocoumarol to human serum albumin (HSA) and alpha 1-acid glycoprotein (alpha 1-AGP) was investigated by affinity chromatography and by combined ultrafiltration (UF) and circular dichroism (CD) methods. For HSA, the ratio of the enantiomeric constants was KR/KS = 2, while for alpha 1-AGP, KS/KR = 3.

Synthesis and conformational studies of poly(L-lysine) based branched polypeptides with Ser and Glu/Leu in the side chains.

In a new group of polypeptides, the branches were composed of DL-Ala oligopeptide, L-serine and L-leucine or L-glutamic acid residues. The synthesis of eight different side-chain combinations is described. In the first group, Ser was attached directly to the epsilon-amino groups of polylysine, and Leu or Glu was situated at the side chain end (poly[Lys(X(i)-DL-Ala(m)-Ser(j))]). Alternatively, Leu or Glu was positioned next to the polylysine backbone (poly[Lys(Ser(j)-DL-Ala(m)-X(i))], where X=L-Leu or L-Glu and m approximately 3-6, i

Influence of side-chain-terminating moieties on the conformation of branched polypeptides and their conjugates with 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone.

Poly(Lys-(Xi-DL-Alam] polypeptides carrying hydrophilic (X = His, Glu, Lys) or hydrophobic (X = Nle, Ile, Phe) amino acid residues and their conjugates with 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone were synthesized. The conformational properties of carrier polypeptides and conjugates were studied by circular dichroism (CD) spectroscopy in the wavelength regions 190-250 and 310-380 nm, with the emphasis on analysis under near physiological conditions. Based on CD studies, it could be demonstrated that the helix-forming capacity appears to be related to the hydrophobic nature of the branch-terminating amino acid of the branched polypeptides. With respect to carrier function, the presence of a coupled derivative of oxazolone at the side chain termini generally promotes the formation of helical secondary structure. The absolute configuration of the side-chain-terminating amino acids was found to be important for the local orientation of the hapten molecule in the conjugates.

Synthesis of N-(phosphonoacetyl)-dipeptide derivatives and evaluation of their antihypertensive activity.

A series of N-(phosphonoacetyl)-dipeptide derivatives was synthesized for pharmacological testing as antihypertensive compounds. Several of these compounds demonstrated a moderate antihypertensive effect in Wistar spontaneous hypertensive rats (SHR) with p.o. dosing. ACE inhibition by the compounds was studied using ACE from rat plasma and lung. Inhibitors containing esterified C-termini are pro-drugs and showed activity only for plasma ACE.

Interaction of the antitumour drug 4'-(9-acridinylamino)-methanesulfon-m-anisidine.HCl (m-AMSA) with nucleic acids.

The interaction of AMSA with nucleic acids was studied by several techniques. Melting temperature and CD studies equally suggest that AMSA-binding is interfering with the secondary structure of DNA. An overlap by two mechanism of binding seems to exist. Based on the CD measurements at low drug concentration intercalation is the most likely way of binding. At higher drug concentration stacking interaction predominates leading to cooperativity and formation of oriented sheets of aromatic ring-systems as reflected in the optical activity induced in the metachromatic band of the achiral drug. No base-pair specificity could be confirmed; however, a high affinity of AMSA to poly(A) chains was demonstrated. The CD measurements did not indicate any significant interaction with RNA. The selectivity of the AMSA-DNA interaction can be regarded as an important argument in favour of the role of this interaction in the anti-tumour effect of the drug.

Carrier design: conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly (L-lysine).

The present study was undertaken to examine the influence of the reversal of the side-chain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L-Lys) backbone and the DL-Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly[Lys-(Xi)] (XK) and poly[Lys-(DL-Alam-Xi)] (AXK) when X = Ala, D-Ala, Leu, D-Leu, Phe, D-Phe, Ile, Pro, Glu, D-Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxy-benzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo(DL-Ala) chains to XK by using N-carboxy-DL-Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L-Lys), poly[Lys-(Xi)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(Xi)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides.

Secondary structure of glycosaminoglycans.

It has been shown by X-ray diffraction and chiroptic spectroscopy that the glycosaminoglycans in connective tissue develop a helical structure; this induces the coordination of polycations and scleroproteins in their environment polycations and promotes or inhibites fibrillary aggregation. Analysis of the ORD and CD spectra of various glycosaminoglycans, as well as those of oversulphated and desulphated preparations allowed the following conclusions concerning the secondary structure. 1. For the helical structure of glycosaminoglycans a chain-length of 120 to 150 A and the presence of at least one sulphate group per two disaccharides are required. The helical structure is not influenced by binding to the protein skeleton, e.g. in proteoglycans. 2. Chiroptical properties of the glycosaminoglycan--either in themselves, or if their methylene blue complexes are studied--are determined by the degree of their sulphatation. 3. The degree of sulphatation determines the secondary structure of the molecule and thus also its biological properties. Fibrillogenesis in vitro and probably also in vivo are determined by the strength of the bond between collagen and glycosaminoglycans, in addition to the effect inducing the coordination. Structural analysis of the polysaccharides supports the coordinated fibrillary structure of GAG, as already assumed on the basis of polarization microscopic methods.

The role of sulphation of glycosaminoglycans in their structural and functional characteristics.

The CD spectra in the far ultraviolet and the CD and ORD spectra of methylene blue-complexes (MB-complex) of glycosaminoglycans (GAG) with different sulphate contents were studied in comparison with their effect on the in vitro formation of collagen fibres. The following polyanions were investigated: Chondroitin sulphate-A with different sulphate contents, oversulphated chondroitin sulphate, heparin and dextran sulphate and their desulphated derivatives. It was observed that the MB-complex of polysaccharides with the same backbone structure, but with different sulphate contents might show ORD and CD spectra of exciton type, but of inverse sign. This phenomenon was interpreted on the basis of different types of aggregation of dye molecules bound to the polysaccharides. The biological effect of GAG changed also with the sulphate content. This suggests that it is the extent of sulphation rather than the glycosidic structure of GAG which determines their chiroptical and functional properties.

Biological significance of helical conformation of acid polysaccharides.

Optical rotary dispersion spectra have been studied of methylene blue complexes of glycosaminoglycans and different derivates of chondroitin 4-sulphate (free and protein bound complexes of different molecular weights and degrees of sulphation). Structural requirements of Ch-4S induction of the extrinsic Cotton effect in methylene blue were formulated. This Cotton effect is supposed to reflect the right-handed helical structure of the polysaccharide molecules. Ch-4S displayed induced Cotton effect both in free and in protein bound form; molecular weight should exceed 2-3000 (at least 4-6 disaccharide units), degree of sulphation should exceed 3% sulphate-sulphur content (more than 0.5 sulphate per one disaccharide unit). For comparison the effect of different derivatives of Ch-4S on the rate of in vitro collagen fibril formation was investigated. Close correlation was found between the macrostructural characteristics and the possible biological effect of Ch-4S. The biological significance of the macrostructural properties of Ch-4S is discussed.

Stereoselective effect of warfarin and bilirubin on the binding of 5-(o-chlorophenyl)-1,3-dihydro-3-methyl-7-nitro-2H-1,4-benzodiazepin-2- one enantiomers to human serum albumin.

The binding of the title benzodiazepine enantiomers and its modulation by warfarin and bilirubin were studied by chromatography on human serum albumin (HSA) immobilized on Sepharose 4B, and also by a combination of ultrafiltration and circular dichroism (UF-CD) methods. In the absence of warfarin and bilirubin the binding of the benzodiazepine was not stereoselective. (S)-Benzodiazepine and (S)-warfarin mutually increased the binding of each other, while the binding of (R)-benzodiazepine was preferentially enhanced on HSA saturated with bilirubin.

Effect of D-amino acid substitution in a mucin 2 epitope on mucin-specific monoclonal antibody recognition.

We have studied the influence of D-amino acid substitution in the flanking region on the antibody recognition of the 19TGTQ22 epitope core in the tandem repeat of mucin 2 (MUC2) glycoprotein. Analogue peptides corresponding to the optimal epitope sequence (16PTPTGTQ22) have been prepared by the replacement of single or multiple L-amino acid residues at the N-terminal part of the molecule. According to previous studies, this portion of the all-L 16PTPTGTQ22 peptide possesses a beta-turn secondary structure important for efficient monoclonal antibody interaction. The binding properties of sequentially modified peptides (pTPTGTQ, ptPTGTQ, ptpTGTQ, and ptptGTQ) have been analyzed by a MUC2 glycoprotein specific monoclonal antibody (MAb 996) using RIA inhibition assay and characterized by IC50 values. At the same time, we have investigated the secondary structure of the compounds by circular dichroism and Fourier transform infrared spectroscopy in solution. Our data showed that the presence of D-amino acid residue(s) at position(s) 16P, 16PT17, or 16PTP18 resulted in gradually decreasing antibody binding, but the replacement of the L-Thr at position 19 almost abolished activity. Parallel with this reduction, changes in the conformer population have been detected. The propensity of the pTPTGTQ peptide to adopt folded, most probably beta-turn, structure in water can be in correlation with its essentially preserved antibody recognition. After further substitution, the peptide still contained beta- and/or gamma-turn folded secondary structural elements. The conformation of peptide ptptGTQ could be characterized mostly by semiextended (polyproline II) and probably classic gamma-turn conformers built up from D residues.

The 90 kDa heat shock protein (hsp90) induces the condensation of the chromatin structure.

The 90 kDa heat shock protein (hsp90) is a member of the "chaperone-complex" of steroid receptors believed to be partially or transiently localized in the cell nucleus. Demonstrating that hsp90 has an ATP binding site and autophosphorylating activity we have observed that histones, especially histone H1, are able to modulate the autophosphorylation of hsp90 [Csermely, P. and Kahn, C.R. (1991) J. Biol. Chem. 266, 4943-4950]. Our present data suggest a direct interaction of hsp90 with histones, showing that hsp90 is able to bind histone-agarose and enhances the binding of histones to DNA. Circular dichroism spectra of rat liver chromatin indicate that hsp90 induces a tighter, condensed state of the chromatin structure which is resistant against disruption by high salt treatment. Interactions of hsp90 with the chromatin may be important in regulating the transcriptional activity of steroid receptors and other transcription factors.

Synthesis, solution conformation and interleukin-6-related activities of interleukin-6 peptides.

Interleukin-6 (IL-6) is a member of the cytokine superfamily characterised by a wide variety of biological activities on various cell types. IL-6 exerts pleiotropic activities on hematopoiesis in the immune response and it is the main regulator of acute-phase protein synthesis in liver cells. According to structure-function studies, residues of helix A located at the N-terminal part and/or helix D of the C-terminal part of the protein are involved in the induction of acute-phase responses. Two groups of synthetic peptides corresponding to the 18-46 N-terminal and the 168-185 C-terminal regions of the IL-6 were prepared by solid-phase synthesis to identify structural requirements for induction of fibrinogen or complement factor B synthesis. These peptides were characterised by amino acid analysis, analytical reversed-phase high-performance liquid chromatography, fast atom bombardment mass spectrometry, and circular dichroism (CD) spectroscopy. CD results showed that under appropriate conditions both 18-46 and 168-185 related peptides are able to adopt markedly ordered conformation. We demonstrated that even octapeptides from the N-terminal part and truncated derivatives of the C-terminal region preserved some tendency to display the CD curve of periodic conformation. The ability of the peptides to induce de novo synthesis of acute-phase proteins was evaluated by measuring fibrinogen and complement factor B levels in the supernatants of human HepG2 cells. These results showed that residues 21-34 are critical for eliciting fibrinogen synthesis in the presence or absence of IL-6. In contrast, the full-length 168-185 peptide is required for the induction of complement factor B response.

Sequential order of T and B cell epitopes affects immunogenicity but not antibody recognition of the B cell epitope.

Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known murine T cell epitope, both in T-B and B-T orientations, show that induction of high murine anti-MUC1 antibody titers is dependent on the presence and orientation of the T cell determinant. However, the sequential order of the epitopes does not affect binding of anti-B cell epitope antibodies to the constructs. Haplotype mismatching leads to a significant lowering of the anti-MUC1 antibody responses, implicating a central role for the T cell epitope in eliciting anti-B cell epitope responses. Secondary structure analysis by circular dichroism spectroscopy reveals the T-B construct to be partially ordered, while the B-T peptide adopts a highly ordered conformation in trifluoroethanol. These studies suggest that the sequential order of epitopes may significantly alter the immunogenicity of the peptide but may not necessarily affect its antigenicity. Immunogenicity of the peptide constructs may be governed by subtle differences in secondary structure, leading to variation in the way peptides are presented or processed within cells governing immune responses. These findings have relevance for the construction of peptides to be utilized as potential synthetic vaccines and for the design of peptide immunogens.

Absolute configuration and local anaesthetic activity of cis and trans 2-dimethylaminomethyl-cyclohexyl benzoates.

All the four stereoisomers of 2-dimethylaminomethyl-cyclohexyl benzoate (enantiomers of 4 and 5) were prepared in optically pure state and their absolute configurations determined by chiroptical methods and genetic correlations. The configurational assignments obtained by the two independent methods were in full agreement and the absolute configuration of the four stereoisomers are (+)-(1S,2S)-4, (-)-(1R,2R)-4, (+)-(1S,2R)-5, and (-)-(1R,2S)-5. The local anaesthetic activity of the four compounds were also determined.

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.

Is amyloid deposition in Alzheimer's disease preceded by an environment-induced double conformational transition?

Fragments of amyloid polypeptide (A4 or beta-protein) were synthesized on solid phase. Circular dichroism (CD) measurements showed that the N-terminal 1-12 fragment assumes an unordered conformation in water, but probably adopts a type I(III) beta-turn in trifluoroethanol (TFE). The central 11-25 fragment has a beta-sheet conformation in aqueous solution, while the full-length N-terminal 1-28 peptide shows helical features in TFE as well as in TFE-water mixtures. Based on secondary structural predictions, molecular mechanical calculations, and the differing and size-dependent conformational propensities of the smaller fragments in aqueous solution, the amyloid peptide is likely to undergo a double conformational transition that is characterized by a pleating process of its central segment. This conformational transition may start spontaneously above a critical peptide concentration, or it may be triggered by age-related or pathological changes ("watering") of the extracellular environment.

Breakage in alpha-helix: a recognition site for anti-rabies virus ribonucleoprotein antibody.

Peptides composed of variants of a B-cell epitope followed by a T-cell determinant of rabies virus ribonucleoprotein (RNP) have been synthesized to elicit antibody production in mice. Conformation of the peptides was characterized by secondary structural prediction and circular dichroism measurements. It was found that only synthetic peptides with disrupted helical structure in the antigenic region were active on immunoblot assay, performed against a natural anti-protein monoclonal antibody (MAb) and provoked virus-neutralizing antibody production.