Tailoring Uptake Efficacy of HSV-1 gD Derived Carrier Peptides.

Regions of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) were chosen to design carrier peptides based on the known tertiary structure of the virus entry receptor complexes. These complexes consist of the following: HSV-1 gD-nectin-1 and HSV-1 gD-herpesvirus entry mediator (HVEM). Three sets of peptides were synthesised with sequences covering the (i) N-terminal HVEM- and nectin-1 binding region -5-42, (ii) the 181-216 medium region containing nectin-1 binding sequences and (iii) the C-terminal nectin-1 binding region 214-255. The carrier candidates were prepared with acetylated and 5(6)-carboxyfluorescein labelled N-termini. The peptides were chemically characterised and their conformational features in solution were also determined. In vitro internalisation profile and intracellular localisation were evaluated on SH-SY5Y neuroblastoma cells. Peptide originated from the C-terminal region 224-247 of the HSV-1 gD showed remarkable internalisation compared to the other peptides with low to moderate entry. Electronic circular dichroism secondary structure studies of the peptides revealed that the most effectively internalised peptides exhibit high helical propensity at increasing TFE concentrations. We proved that oligopeptides derived from the nectin-1 binding region are promising candidates-with possibility of Lys237Arg and/or Trp241Phe substitutions-for side-reaction free conjugation of bioactive compounds-drugs or gene therapy agents-as cargos.

Photochemical and Structural Studies on Cyclic Peptide Models.

Ultra-violet (UV) irradiation has a significant impact on the structure and function of proteins that is supposed to be in relationship with the tryptophan-mediated photolysis of disulfide bonds. To investigate the correlation between the photoexcitation of Trp residues in polypeptides and the associated reduction of disulfide bridges, a series of small, cyclic oligopeptide models were analyzed in this work. Average distances between the aromatic side chains and the disulfide bridge were determined following molecular mechanics (MM) geometry optimizations. In this way, the possibility of cation⁻π interactions was also investigated. Molecular mechanics calculations revealed that the shortest distance between the side chain of the Trp residues and the disulfide bridge is approximately 5 Å in the cyclic pentapeptide models. Based on this, three tryptophan-containing cyclopeptide models were synthesized and analyzed by nuclear magnetic resonance (NMR) spectroscopy. Experimental data and detailed molecular dynamics (MD) simulations were in good agreement with MM geometry calculations. Selected model peptides were subjected to photolytic degradation to study the correlation of structural features and the photolytic cleavage of disulfide bonds in solution. Formation of free sulfhydryl groups upon illumination with near UV light was monitored by fluorescence spectroscopy after chemical derivatization with 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM) and mass spectrometry. Liquid cromatography-mass spectrometry (LC-MS) measurements indicated the presence of multiple photooxidation products (e.g., dimers, multimers and other oxidated products), suggesting that besides the photolysis of disulfide bonds secondary photolytic processes take place.

Structural insight into a partially unfolded state preceding aggregation in an intracellular lipid-binding protein.

Human ileal bile acid-binding protein (I-BABP) has a key role in the intracellular transport and metabolic targeting of bile salts. Similar to other members of the family of intracellular lipid-binding proteins (iLBPs), disorder-order transitions and local unfolding processes are thought to mediate ligand entry and release in human I-BABP. To gain insight into the stability of various protein regions, the temperature response of human I-BABP was investigated using NMR, CD and fluorescence spectroscopy, as well as molecular dynamics (MD) simulations. A joint analysis of NMR thermal melting and relaxation dispersion data indicates a complex pattern of internal dynamics with a dominating single barrier and a likely presence of rapidly exchanging conformational substates on both sides of the barrier. Moreover, our residue-specific analysis uncovers a partially unfolded U* state in which part of the helical region with three proximate ß-strands contains a substantial amount of residual structure, whereas several segments of the C-terminal half exhibit a high susceptibility to temperature elevation. Cluster analysis of atomic temperature responses indicates a thermodynamic coupling between distant protein sites including the bottom of the ß-barrel, the E-F region and part of the helical cap. MD simulations up to 1 µs show correlated motions in the same protein regions and together with the NMR data suggest a role for the highly dynamic D-E turn and E-F region in the initiation of unfolding. The response of human I-BABP to temperature elevation is discussed in the context of the folding/unfolding behaviour of different members of the iLBP family.

Characterization of desmoglein-3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy, cytotoxicity, stability, and their conformational features.

Desmoglein-3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full-length peptide (Dsg3/189-205, Dsg3/206-222, Dsg3/342-358, and Dsg3/761-777) and its N-terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure-activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)-γ content of the supernatants was measured by enzyme-linked immunosorbent assay. In the in vitro conditions, peptides were stable and non-cytotoxic. The in vitro IFN-γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342-358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192-205, Dsg3/763-777, and Dsg3/764-777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors.

Development of cyclic NGR peptides with thioether linkage: structure and dynamics determining deamidation and bioactivity.

NGR peptides that recognize CD13 receptors in tumor neovasculature are of high interest, in particular due to their potential applications in drug targeting. Here we report the synthesis and structural analysis of novel thioether bond-linked cyclic NGR peptides. Our results show that their chemostability (resistance against spontaneous decomposition forming isoAsp and Asp derivatives) strongly depends on both sample handling conditions and structural properties. A significant correlation was found between chemostability and structural measures, such as NH(Gly)-CO(Asn-sc) distances. The side-chain orientation of Asn is a key determining factor; if it is turned away from HN(Gly), the chemostability increases. Structure stabilizing factors (e.g., H-bonds) lower their internal dynamics, and thus biomolecules become even more resistant against spontaneous decomposition. The effect of cyclic NGR peptides on cell adhesion was examined in A2058 melanoma cell lines. It was found that some of the investigated peptides gradually increased cell adhesion with long-term characteristics, indicating time-dependent formation of integrin binding isoAsp derivatives that are responsible for the adhesion-inducing effect.

Calcium carbonate crystal growth beneath Langmuir monolayers of acidic ß-hairpin peptides.

Four amphiphilic peptides with designed hairpin structure were synthesized and their monolayers were employed as model systems to study biologically inspired calcium carbonate crystallization. Langmuir monolayers of hairpin peptides were investigated by surface pressure area isotherms, surface potential isotherms, Brewster angle microscopy (BAM), atomic force microscopy (AFM) and Fourier transform infrared (FTIR) spectroscopy. A ß-hairpin conformation was found for all peptides at the air-water interface although their packing arrangements seem to be different. Crystallization of calcium carbonate under these peptide monolayers was investigated at different surface pressures and growth times both by in situ optical microscopy, BAM and ex situ investigations such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). An amorphous calcium carbonate precursor was found at the initial crystallization stage. The crystallization process occurred in three stages. It starts from the nucleation of amorphous particles being a kinetically controlled process. Crystal nuclei subsequently aggregate to large particles and vaterite crystals start to form inside the amorphous layer, with the monolayer fluidity exerting an important role. The third process includes the re-crystallization of vaterite to calcite, which is thermodynamically controlled by monolayer structural factors including the monolayer flexibility and packing arrangement of the polar headgroups. Thus, the kinetic factors, monolayer fluidity and flexibility as well as structure factors govern the crystal morphology and polymorph distribution simultaneously and synergistically.

Comprehensive chiroptical study of proline-containing diamide compounds.

The continuously growing interest in the understanding of peptide folding led to the conformational investigation of methylamides of N-acetyl-amino acids as diamide models. Here we report the results of detailed conformational analysis on Ac-Pro-NHMe and Ac-ß-HPro-NHMe diamides. These compounds were analyzed by experimental and computational methods, the conformational distributions obtained by Density Functional Theory (DFT) calculations for isolated and solvated diamide compounds are discussed. The conformational preference of proline-containing diamide compounds as a function of the ambience was observed by a number of chiroptical spectroscopic techniques, such as vibrational circular dichroism (VCD), electronic circular dichroism (ECD), Raman optical activity (ROA) spectroscopy, and additionally by single crystal X-ray diffraction analyses. Based on a comparison between Ac-Pro-NHMe and Ac-ß-HPro-NHMe, one can conclude that due to the greater conformational freedom of the ß-HPro derivative, Ac-ß-HPro-NHMe shows different behavior in solid- and solution-phase, as well. Ac-ß-HPro-NHMe tends to form cis Ac-ß-HPro amide conformation in water, dichloromethane, and acetonitrile in contrast to its α-Pro analog. On the other hand, the crystal structure of the ß-HPro compound cannot be related to any of the conformers obtained in vacuum and solution while the X-ray structure of Ac-Pro-NHMe was identified as tα(L)-, which is a trans Ac-Pro amide containing conformer also predominant in polar solvents.

Fluorescence lifetime distributions report on protein destabilisation in quenching experiments.

Tryptophan is the most often investigated intrinsic fluorophore due to its abundance in proteins and its sensitivity to different environmental conditions. Fluorescence quenching is a powerful method to study proteins and acrylamide is a frequently applied quencher in these investigations. Quenching experiments are sometimes distorted by the undesired protein-quencher interactions that can result in a misinterpretation of the results. Here we focused on the identification of the possible side-effects of acrylamide applying fluorescence lifetime measurements. To provide reference data for protein denaturation the fluorescence parameters were also recorded in the presence of different concentrations of guanidine hydrochloride. In circular dichroism experiments we characterized directly the acrylamide effect on the tertiary structure of the proteins. According to the obtained data in experiments with seven tryptophan-containing proteins the full width at half maximum (FWHM) of the fluorescence lifetime distribution is an appropriate parameter to monitor the undesired effects of acrylamide on the proteins.

New m-calpain substrate-based azapeptide inhibitors.

Calpains are intracellular cysteine proteases with several important physiological functions. Calpain inhibitors may be promising tools in the analysis of the function of the enzyme in diseases caused by overexpression/activation. Here, we report on the synthesis, solution conformation, and characterization of novel group of azapeptides whose sequences originate from an efficient m-calpain substrate, TPLKSPPPSPR, described by us earlier and possess varying levels of calpain inhibition. The Lys residue at P1 position was replaced with azaglycine (NH2 -NH-COOH) and further changes were made as follows: the N-terminal or/and C-terminal were truncated, amino acids were also changed at P3, P2, P'1, or P'2 positions. Our results indicate that the identity of amino acid moieties between P4 and P'5 positions is essential for the inhibitory activity. Only changes at position P3 (Pro) are tolerated. Azapeptide analogs, described in this communication could be considered as useful set of compounds for elucidation of the enzyme interaction at P and P' sites.

Binding of new cationic porphyrin-tetrapeptide conjugates to nucleoprotein complexes.

Ongoing research on DNA binding of cationic porphyrin derivatives and their conjugates is a subject of growing interest because of their possible DNA binding and demonstrated biological properties. In this study nucleoprotein binding of tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin (TMPCP) and tetrapeptides conjugated TMPCP (TMPCP-4P) and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin (BMPCP-4P2) was investigated with comprehensive spectroscopic methods. The key observation is that tetrapeptide-conjugates of cationic porphyrins with two or three positive charges bind to encapsidated DNA in T7 phage nucleoprotein complex. The binding modes were analyzed by fluorescent energy transfer, fluorescent life time and CD measurements. Intercalative binding is most feasible when tricationic ligands complex with DNA, especially when it is in close connection with protein capsid. It was found that larger ligand BMPCP-4P2 binds externally to encapsidated T7 DNA, and complex externally as well as by intercalation when the DNA accommodate to relaxed B-conformation. In the case of TMPCP and TMPCP-4P the intercalation is the predominant binding form both in nucleoprotein (NP) and preheated complexes. Further, melting experiments revealed that bound porphyrins do not influence the capsid stability or protein-DNA interactions, but efficiently stabilize the double helical structure of DNA without respect to binding form. A good correlation was found between porphyrin/base pair ration and DNA strand separation temperature.

Role of N- or C-terminal biotinylation in autoantibody recognition of citrullin containing filaggrin epitope peptides in rheumatoid arthritis.

Here, we report on the synthesis, conformational analysis, and autoantibody binding properties of new sets of rheumatoid arthritis (RA) specific biotin-peptide conjugates derived from filaggrin epitope peptides. The biotin with or without a linker was attached to the Cit or Arg containing epitope core ((311)TXGRS(315)) or epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) peptide (where X = Cit), through an amide bond at the N- or C-terminal of the epitopes. Antibody binding was detected by indirect enzyme-linked immunosorbent assay (ELISA) using sera from RA, Systemic lupus erythematosus (SLE) patients, as well as healthy individuals, and the secondary structure of conjugates was investigated by electronic circular dichroism (ECD). We found that autoantibodies from RA patients recognize specifically both filaggrin epitope region ((306)SHQESTXGXSXGRSGRSGS(324)) and short epitope core ((311)TXGRS(315)) peptides. Our data also indicate that the positioning of the biotin label within a peptide sequence can markedly influence the antibody binding, but the length of the linker incorporated has essentially no effect on the recognition. ECD experiments demonstrate that the Arg/Cit change does not influence the solution conformation of the peptide conjugates. However, the presence and position of the biotin moiety has a pronounced effect on the conformation of the 5-mer epitope core peptides, while it does not alter the secondary structure of the 19-mer epitope region peptides.

Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676, and the effect of D-Asp and iso-Asp substitution at Asp672 residue.

Amyloid precursor protein (APP) fragment containing amino acids 667-676, (APP667₋676), is a substrate for ß-secretase which is responsible for generating amyloid ß peptides. Conformational analysis of APP667₋676 peptide [Ac-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH2] and the effect of substitution of Asp672 with D-Asp and iso-L-Asp, studied for the first time, demonstrate that the peptide backbone of APP667₋676 is flexible and adopts different conformations in different solvent environments (water, trifluoroethanol and dimethylsulfoxide). A major conformational difference was observed in trifluoroethanol solvent when Asp672 is substituted with D-Asp and iso-Asp. These conformational changes involved in APP667₋676 may assist in understanding the interactions between ß-secretase and APP667₋676, with relevance to Alzheimer's disease.

Syntheses and DNA binding of new cationic porphyrin-tetrapeptide conjugates.

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet. We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins. Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.

Conformational studies on chiral rhodium complexes by ECD and VCD spectroscopy.

This article reports vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopic studies in acetonitrile on the chiral Rh(2)(O-Phe-Cbz)(1)(OAc)(3) and Rh(2)(O-Phe-Ac)(1)(OAc)(3) complexes (abbreviated Rh(2)Z(1) and Rh(2)Ac(1) , respectively; Phe, L-phenylalanine; Cbz, benzyloxycarbonyl; Ac, acetyl) supported by theoretical calculations. The ECD spectra of the complexes depend on temperature that indicates the conformational mobility of the chiral ligands. Calculations of the VCD spectra were performed at ab initio (DFT) level of theory using Gaussian 03 [B3LYP functional combined with the LANL2DZ basis set for the dirhodium core and the 6-31G(d) basis set for other atoms]. The population-weighted sums of the computed VCD spectra of the conformers are in excellent agreement with the experimental VCD spectra. The combination of the VCD and ECD spectroscopic methods led us to the structural characterization of the complexes.

Vibrational and chiroptical spectroscopic characterization of gamma-turn model cyclic tetrapeptides containing two beta-Ala residues.

The optical spectroscopic characterization of gamma-turns in solution is uncertain and their distinction from beta-turns is often difficult. This work reports systematic ECD and vibrational circular dichroism (VCD) spectroscopic studies on gamma-turn model cyclic tetrapeptides cyclo(Ala-beta-Ala-Pro-beta-Ala) (1), cyclo(Pro-beta-Ala-Pro-beta-Ala) (2) and cyclo(Ala-beta-Ala-Ala-beta-Ala) (3). Conformational analysis performed at the 6-31G(d)/B3LYP level of theory using an adequate PCM solvent model predicted one predominant conformer for 1-3, featuring two inverse gamma-turns. The ECD spectra in ACN of 1 and 2 are characterized by a negative n-->pi* band near 230 nm and a positive pi-->pi* band below 200 nm with a long wavelength shoulder. The ECD spectra in TFE of 1-3 show similar spectra with blue-shifted bands. The VCD spectra in ACN-d(3) of 1 and 2 show a +/-/+/- amide I sign pattern resulting from four uncoupled vibrations in the case of 1 and a sequence of two positive couplets in the case of 2. A -/+/+/- amide I VCD pattern was measured for 3 in TFE-d(2). All three peptides give a positive couplet or couplet-like feature (+/-) in the amide II region. VCD spectroscopy, in agreement with theoretical calculations revealed that low frequency amide I vibrations (at approximately 1630 cm(-1) or below) are indicative of a C(7) H-bonded inverse gamma-turns with Pro in position 2, while gamma-turns encompassing Ala absorb at higher frequency (above 1645 cm(-1)).

Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies.

Antifreeze glycoproteins enable life at temperatures below the freezing point of physiological solutions. They usually consist of the repetitive tripeptide unit (-Ala-Ala-Thr-) with the disaccharide alpha-D-galactosyl-(1-3)-beta-N-acetyl-D-galactosamine attached to each hydroxyl group of threonine. Monoglycosylated analogues have been synthesized from the corresponding monoglycosylated threonine building block by microwave-assisted solid phase peptide synthesis. This method allows the preparation of analogues containing sequence variations which are not accessible by other synthetic methods. As antifreeze glycoproteins consist of numerous isoforms they are difficult to obtain in pure form from natural sources. The synthetic peptides have been structurally analyzed by CD and NMR spectroscopy in proton exchange experiments revealing a structure as flexible as reported for the native peptides. Microphysical recrystallization tests show an ice structuring influence and ice growth inhibition depending on the concentration, chain length and sequence of the peptides.

The role of nanoparticle concentration-dependent induction of cellular stress in the internalization of non-toxic cationic magnetoliposomes.

Magnetoliposomes (MLs), built up of ultrasmall iron oxide cores each individually surrounded by a lipid bilayer, have emerged as highly biocompatible nanoparticles and promising tools in many biomedical applications. To improve cell uptake, cationic amphiphiles are inserted into the ML coat, but this often induces cytotoxic effects. In the present work, we synthesized and tested a cationic peptide-lipid conjugate (dipalmitoylphosphatidylethanolamine-succinyl-tetralysine [DPPE-succ-(Lys)4]) which is entirely composed of biodegradable moieties and specifically designed to exert minimal cytotoxic effects. Uptake studies with both murine 3T3 fibroblasts and C17.2 neural progenitor cells shows 95.63 +/- 5.83 pg Fe and 87.46 +/- 5.62 pg Fe per cell after 24 h, respectively, for 16.66% DPPE-succ-(Lys)4-containing MLs, with no effect on cell viability. However, these high intracellular nanoparticle concentrations transiently affect actin cytoskeleton architecture, formation of focal adhesion complexes and cell proliferation, returning to control levels after approximately 7 days post ML-incubation in both cell types. This study points out the great need for thorough characterization of cell-nanoparticle interactions as subtle time-dependent effects are hard to monitor and commonly used viability and functionality assays are not sufficient to address the broad spectrum of possible interferences of the nanoparticle with normal cell functioning.

Role of structure-proteins in the porphyrin-DNA interaction.

We studied the complexation of meso-tetrakis(4-N-methylpyridyl)porphyrin (TMPyP) with HeLa nucleosomes and compared it to our earlier results on T7 phage nucleoprotein complex (NP) and isolated DNA. To identify binding modes and relative concentrations of the bound TMPyP forms, the porphyrin absorption spectra were analyzed at various base pair/porphyrin ratios. Spectral decomposition and circular dichroism measurements proved that the two main binding modes of TMPyP, i.e., external binding and intercalation occur also in the nucleosomes. The DNA superstructure maintained by the proteins decreases its accessibility for TMPyP similarly in both nucleoproteins. A difference is observed between the partitioning of the two binding modes: in the case of nucleosome the ratio of intercalation to groove-binding is changed from 60/40 to 40/60 as determined for T7 NP and for isolated DNA-s. Using UV and CD melting studies, we revealed that TMPyP destabilizes the DNA-protein interaction in the nucleosomes but not in the T7 phage. Lastly, photoinduced reaction of bound TMPyP caused alterations in DNA structures and DNA-protein interactions within both nucleoprotein complexes; the nucleosomes were found to be more sensitive to the photoreaction.

Synthesis and antibody recognition of synthetic antigens from MUC1.

In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.