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Donnees scientifiques produites : https://portuslimen.eu/https://amidex.hypotheses.org/9931 Introduction La caracterisation de materiaux de construction et leur provenance sont generalement etudiees par des analyses petrographiques, optiques, mineralogiques et geochimiques. Efficace, rapide, non-destructive et dans certains cas plus precise que les techniques conventionnelles, la Spectroscopie Moyen Infrarouge (SMIR) a Transformee de Fourier (FTIR) est une approche alternative pour etudier le...

International audience

Molluscs defend themselves against predation and environmental stressors through the possession of mineralized shells. Mussels are widely used to predict the effects of abiotic factors such as salinity and pH on marine calcifiers in the context of changing ocean conditions. Shell matrix proteins are part of the molecular control regulating the biomineralization processes underpinning shell production. Under changing environmental conditions, differential expression of these proteins leads to the phenotypic plasticity of shells seen in many mollusc species. Low salinity decreases the availability of calcium and inorganic carbon in seawater and consequently energetic constraints often lead to thin, small and fragile shells in Mytilid mussels inhabiting Baltic Sea. To understand how the modulation of shell matrix proteins alters biomineralization, we compared the shell proteomes of mussels living under full marine conditions in the North Sea to those living in the low saline Baltic Sea. Modulation of proteins comprising the Mytilus biomineralization tool kit is observed. These data showed a relative increase in chitin related proteins, decrease in SD-rich, GA-rich shell matrix proteins indicating that altered protein scaffolding and mineral nucleation lead to impaired shell microstructures influencing shell resistance in Baltic Mytilid mussels. Interestingly, proteins with immunity domains in the shell matrix are also found to be modulated. Shell traits such as periostracum thickness, organic content and fracture resistance qualitatively correlates with the modulation of SMPs in Mytilid mussels providing key insights into control of biomineralization at molecular level in the context of changing marine conditions., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; The use of ochre in mortuary practices was widespread during prehistory. In northern Thailand, several painted rock-shelters attributed to Iron age or Neolithic cultures have provided burials characterized by the presence of pigment fragments and ochres deposits. In the case of Doi Pha Kan, data collected during excavations have highlighted the important role of ochre in funerary rites dating from 13,172 to 10,986 cal BC. The presence of painted panels overhanging Hoabinhian burials raises the question of their chronology and association to painting activities. To investigate this issue at Doi Pha Kan, we applied a multi-analytical approach (optical microscopy, SEM-EDS, Raman spectroscopy, XRF spectroscopy, XRD and μXRD) on painting pigments and ochres associated with a grave (dpk E-5) and the archaeological context to determine the mineralogical and elemental composition of pigment raw materials. We showed that the composition of raw pigments from the archaeological context is homogeneous, suggesting a common geological source characterized by the presence of As. In contrast, pigments on the painted panel have variable compositions indicating that panels were created in different time periods. The pigment used for anthropomorphic figure is the only one similar to the materials identified in the archaeological levels and burial at Doi Pha Kan. Without possibility of direct dating, it is not possible to establish a direct link between the these anthropomorphic figure paintings and mortuary practices, however two painted wall fragments were discovered in the archaeological layers attest to a long tradition of rock painting for at least 13,000 years at this site.

En amont de Vierzon, le systeme fluviatile fossile du Cher est compose de sept nappes alluviales, quatre formations sableuses etagees sur le versant ouest de la vallee et trois formations emboitees dans le fosse tectonique dans lequel coule actuellement la riviere. D’apres les donnees geochronologiques (ESR) disponibles, ces formations se sont deposees entre environ 1 Ma et 60 ka. Le site acheuleen de la Noira (Brinay, Cher) est situe a mi-hauteur du versant ouest, a la base de la nappe alluviale de la formation des Fougeres. A la Noira, l’unite grossiere de base (Unite a), deposee pres du versant et recouverte par la Formation des Fougeres, correspondrait a une phase de transition interglaciaire-glaciaire. Les hommes y ont exploite les plaques de meulieres contenues dans des sediments grossiers deposes par solifluxion sur le substratum apres l’incision du Cher. Les fragments des plaques qu’ils ont brisees ont ete faconnes en bifaces et ont servi au debitage d’eclats. Cette Unite a et les ateliers qu’elle renfermait ont ensuite ete recouverts par des colluvions, puis partiellement cryoturbes avant le recouvrement par la puissante formation fluviatile sableuse des Fougeres. A la Noira, est constituee, sur plus de 6 m d’epaisseur, de trois unites sableuses (Unites b, c et d) recoupes par plusieurs discontinuites, recouvertes par des depots pente, des debris cryoclastes, ou soulignees par des fentes de gel. D’apres les dates ESR (âge moyen pondere de 665 ± 55 ka), la formation se serait deposee apres l’unite de base a, dans la premiere partie du MIS 16.

International audience; Bones mostly consist of composite materials based on almost equivalent volume fractions of mineral (apatite) and organic (collagen) components. Accordingly, their infrared spectroscopic properties should reflect this composite nature. In this letter, we show by theory and experiment that the variability of the strong phosphate bands in the ATR-FTIR spectra of a series of modern and archeological bone samples can be related to electrostatic interactions affecting apatite particles and depending on the bone collagen content. Key parameters controlling the shape of these bands are the mineral volume fraction and the dielectric constant of the embedding matrix. The magnitude of these effects is larger than the one related to microscopic changes of the apatite structure. Consequently, the interplay of microscopic and macroscopic parameters should be considered when using FTIR spectroscopy to monitor the preservation state of bioapatite during diagenetic and fossilization processes, especially during the degradation of the organic fraction of bone.

National audience; Dans la vallée du Cher (région Centre-Val de Loire, France), les recherches menées depuis 2003 ont permis d’élaborer un cadre géologique, chronologique et paléoenvironnemental pour les systèmes fluviatiles et les sites préhistoriques associés. A Brinay (Cher), les alluvions sableuses de la nappe des Fougères qui ont recouvert le site acheuléen de la Noira ont été datées par la méthode ESR de 665 ± 55 ka (MIS 16/15). Sur ce site, les études géoarchéologiques et les fouilles montrent que les homininés ont prospecté des amas contenant des matériaux variés issus d’une formation plio-pléistocène plus ancienne et descendus depuis l’interfluve sur le versant (Sous-unité a1/Unité a). Leur stratégie d’approvisionnement semble avoir consisté à récupérer, après tri, des plaques de meulière en éliminant celles qui étaient altérées ou gelées antérieurement. Les plaques ont été brisées et les fragments utilisés comme supports pour le débitage d’éclats et le façonnage de bifaces. Les fouilles ont confirmé la position primaire de ces artefacts dont l’état de fraîcheur est remarquable.La présence des hommes près de la rivière serait donc en partie liée à ces matériaux accessibles. Les meulières recherchées sont des silicifications qui ont été mises au jour lors de la phase d’incision du Cher puis sont descendues dans des coulées sur la pente. Ces phénomènes liés à la cyclicité climatique quaternaire correspondent à une phase de transition en début glaciaire, avant le pléniglaciaire durant lequel les zones non protégées de l’Unité a et du plancher d’incision ont été cryoturbées.

International audience; Tabon Cave is a key site for the understanding of modern human dispersals in the Philippine archipelago and Island Southeast Asia. Nestled in the karst landscape that borders the southwestern coast of Palawan, it has delivered the earliest confirmed Homo sapiens remains in the Philippines dating to the late Pleistocene, as far back as around 47 ka. Among other methods, the broad characteristics of the environment in which these humans once lived may be drawn using stable isotope analysis of the rich guano deposits in the cave, an approach that follows a growing number of studies indicating the potential of guano as a palaeoenvironmental archive. δ13C values reveal the general prevalence of C3 forest tempered by savannah woodland with grassland contributions both well before and slightly after a securely-dated fireplace at 32 ka; the lower interval would refer to OIS 3 or older interglacial periods, while the upper interval would refer to the transition either before or after the Last Glacial Maximum. No useful conclusions are drawn from δ15N results due to suspected ammonia fractionation. Pending future dating efforts for confirmation, this preliminary study contributes to the development of an alternative and promising palaeoenvironmental proxy and hopes to shed further light on the prehistoric odysseys that took place across Island Southeast Asia.

International audience; The Acheulean site of la Noira was fossilized beneath the Fougères fluviatile formation dated by the ESR method at 655 ± 55 ka. Coarse deposits were left at the base after the end of the Cher incision and served as raw material deposits for hominins. The occupation level has yielded bifaces, cores and a flake assemblage in lacustrine millstone. The systematic recording of about 6 500 lithic objects brought to light the reasons underlying the presence of the diverse materials, and elucidated hominin choice of lacustrine millstone for knapping flakes and shaping bifacial pieces. The systematic study of all the materials enabled us to advance hypotheses concerning some of the behaviour identified at the site: sorting of raw materials affected by frost, cursory peripheral knapping of slabs of up to a metre long with hammers weighing several kilogrammes, breaking up of large slabs over 10 cm long and displacement of the fragments. This analysis assesses hominin behaviour and the management and exploitation of raw material deposits at 700 ka.

Expensive and time-consuming preparation procedures for radiocarbon and stable isotope analyses can be conducted on archaeological bone samples even if no collagen is preserved. Such unsuccessful preparation can lead to the partial destruction of valuable archaeological material. Establishing a rapid prescreening method for evaluating the amount of bone collagen while minimizing the impact of sampling constitutes a challenge for the preservation of archaeological collections. This study proposes and discusses a new methodology to detect and quantify collagen content in archaeological bone samples by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. A total of 42 Pleistocene to modern bone samples were selected according to their nitrogen content measured using an elemental analyzer. Comparison of collagen content estimation using ATR-FTIR and mass spectrometry reveals that some of the studied samples are contaminated by a nitrogen source coming from the burial environment. Two different FTIR calibration approaches were tested on the uncontaminated samples: peak-to-peak ratio and multivariate regression (PLS). The two approaches yield similar results with a good correlation of ATR-FTIR analyses and N wt% from 0.7 to 4wt% (R²=0.97–0.99; standard error of estimation ±0.22 to 0.25wt%). While collagen content remains difficult to detect in poorly preserved bones (less than ~3wt%), ATR-FTIR analysis can be a fast alternative for sample screening to optimize the sampling strategy and avoid partial destruction of valuable samples that do not contain enough collagen for further analysis.

The prehistoric site of la Noira, located in the Cher Valley, a tributary of the Loire River in the center of France, has yielded a lithic assemblage composed of large bifacial tools, cores and flakes. The archaeological level, lying on the Tertiary lacustrine limestone bedrock, was covered and fossilized by a 6 m-thick fluvial sandy formation. The mean age value of ESR dates obtained on bleached fluvial quartz grains sampled in the sandy levels covering the archeological level is 665 ± 55 ka, confirming the antiquity of the archaeological assemblage. ESR dates and the technical characteristics of the assemblage suggest that it is among the oldest sites with bifacial technology in Western Europe. Since 2011, following geological and geochronological studies, the archaeological level has been excavated over a surface of about 100 m2. The aim of this paper is to provide new data on the lithic assemblage and to place the lithic patterns of the site in the European technological framework. La Noira is a workshop site, belonging to a key-period of time with the earliest evidence of the bifacial technology in Europe (as for instance levels P–Q of Arago in France or Notarchirico in Italy) contemporaneous with 800–500 ka sites without bifacial technology, such as Happisburgh, Pakefield in England or Isernia in Italy. This phase predates the wide-scale dissemination of the bifacial technology all over Western Europe from the MIS 12. Technological comparisons between these assemblages and a discussion of the diversity of assemblages and technological features point to early episodic arrivals of new traditions in Europe against a background of earlier traditions.

The antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by two transmembrane receptor subunits, IFNAR1 and IFNAR2. To elucidate the role of IFNAR1 in IFN binding and the establishment of biological activity, specific residues of IFNAR1 were mutated. Residues (62)FSSLKLNVY(70) of the S5-S6 loop of the N-terminal subdomain of IFNAR1 and tryptophan-129 of the second subdomain of IFNAR1 were shown to be crucial for IFN-alpha binding and signaling and establishment of biological activity. Mutagenesis of peptide (278)LRV in the third subdomain shows that these residues are critical for IFN-alpha-induced biological activity but not for ligand binding. These data, together with the sequence homology of IFNAR1 with cytokine receptors of known structure and the recently resolved NMR structure of IFNAR2, led to the establishment of a three-dimensional model of the human IFN-alpha/IFNAR1/IFNAR2 complex. This model predicts that following binding of IFN to IFNAR1 and IFNAR2 the receptor complex assumes a "closed form", in which the N-terminal domain of IFNAR1 acts as a lid, resulting in the activation of intracellular kinases. Differences in the primary sequence of individual IFN-alpha subtypes and resulting differences in binding affinity, duration of ligand/receptor association, or both would explain differences in intracellular signal intensities and biological activity observed for individual IFN-alpha subtypes.

A simple method for predicting residues involved in protein interaction sites is proposed. In the absence of any structural report, the procedure identifies linear stretches of sequences as "receptor-binding domains" (RBDs) by analysing hydrophobicity distribution. The sequences of two databases of non-homologous interaction sites eliciting various biological activities were tested; 59-80 % were detected as RBDs. A statistical analysis of amino acid frequencies was carried out in known interaction sites and in predicted RBDs. RBDs were predicted from the 80,000 sequences of the Swissprot database. In both cases, arginine is the most frequently occurring residue. The RBD procedure can also detect residues involved in specific interaction sites such as the DNA-binding (95 % detected) and Ca-binding domains (83 % detected). We report two recent analyses; from the prediction of RBDs in sequences to the experimental demonstration of the functional activities. The examples concern a retroviral Gag protein and a penicillin-binding protein. We support that this method is a quick way to predict protein interaction sites from sequences and is helpful for guiding experiments such as site-specific mutageneses, two-hybrid systems or the synthesis of inhibitors.

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.

Novel analogues of the cell-penetrating peptides penetratin and transportan were synthesized. The distribution of the biotin-labeled peptides in Bowes melanoma cell line has been investigated by indirect fluorescence with fluorescein-streptavidin detection. The time course of uptake of (125)I-labeled transportan analogues has been characterized in the same cell line. Molecular modeling was used to analyze the penetration and the orientation of molecules in a simulated biological membrane. The results, both from molecular modeling and fluorescence studies, imply that penetratin and transportan do not enter the cells by related mechanisms and that they do not belong to the same family of translocating peptides.

Using a molecular modeling method, different conformations of surfactin at a hydrophobic/hydrophilic interface are established. Two conformations of the peptide ring (S1 and S2) provided by NMR experiments built with three different aliphatic chains in folded or extended configurations were studied. For the structures including the S2 peptide ring conformation, the theoretical interfacial molecular area corresponds to the experimental limiting area A0 value obtained with a Langmuir film balance. The peptide ring is positioned in the plane of the interface with the two acidic chains close to each other and protruding in the aqueous phase, and the β-hydroxy fatty acid chain, folded to interact mainly with the Leu2 side chain and also with the Val4 side chain. This design has the largest calculated molecular area and would correspond to the most stable amphipathic structure representing the surfactin experimental behavior in weak compression.

Monoclonal antibodies (mAbs) were produced against gastric H,K-ATPase using a theoretical and experimental strategy based on prediction of linear epitopes by molecular modelling followed by production of anti-peptide antibodies. By analysing the alpha subunit sequence, we predicted several epitopes corresponding to amino acids K519-L533, E543-Y553 and S786-L798 and produced monoclonal antibodies HK519, HK543 and HK786. All three react against gastric H,K-ATPase in RaLISA, immunohistochemistry and Western blots demonstrating that they recognize the native and the SDS-denatured ionic pump and that the epitopes are located at the surface of the native ATPase. Antibody Kd are in the range 6-10x10(-8) M. Monoclonal antibody HK519 is a competitive inhibitor of ATP, in agreement with ATP binding to K519. Neither mAb 543, nor mAb 786 inhibit the ATPase activity. Monoclonal antibody 95111, whose epitope is mapped between residues C529 and E561, competes with mAb HK543 but not with the other two. We suggest that the 95111 epitope is overlapping or very close to the HK543-553 sequence. Induction of E1 conformer by binding FITC to K519 increases the number of mAb 95111 and mAb HK543 epitopes but not that of mAb 786, supporting the fact that the fragment E543-Y553 changes accessibility, maybe during the E1-E2 transconformation.

International audience; 2-D and 3-D NMR techniques were used to investigate the conformations in solution of several peptides and proteins for which crystalline structures are not available yet. Insect defensin A is a small (40 aa) antibiotic protein exhibiting a characteristic 'loop-helix-beta-sheet' structure. A striking analogy was found with charybdotoxin, a scorpion toxin in which a CSH (cysteine stabilized alpha-helix) motif is also present. Wheat phospholipid transfer protein (PLTP) (90 aa) has a 3-D structure resulting from the packing of four helices and of a C-terminal less well-defined fragment. Preliminary results show that PLTP forms a complex with lyso-PC and that such an interaction results in a conformational change affecting principally the C-terminal half of the protein. A last example is given with surfactin, a lipopeptide biosurfactant from bacterial origin. Its protonated form shows a very compact structure in which the two acidic residues located on the top of a 'horse saddle' topology face each other, whereas the ionized form could adopt a more extended conformation. A common property of these compounds is their capacity to interact with lipids. The present structural data open the way for a further establishment of structure-activity relationships.

International audience; A 500 MHz 2D 1H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities, 3JNH-alpha H coupling constants as well as 1H/2H exchange rates and delta delta/delta T temperature coefficients of NH protons strongly support the existence of an alpha-helix (residues 14-24) and of an antiparallel beta-sheet (residues 27-40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from 3JNH-alpha H coupling constants, (iii) 12 hydrogen bonds mostly deduced from 1H/2H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a beta-sheet is linked to an alpha-helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.

Butenafine ( N -4- tert -butylbenzyl- N -methyl-1-naphtalenemethylamine hydrochloride) is an antifungal agent of the benzylamine class that has excellent therapeutic efficacy and a remarkably long duration of action when applied topically to treat various mycoses. Given the lipophilic nature of the molecule, efficacy may be related to an interaction with cell membrane phospholipids and permeabilization of the fungal cell wall. Similarly, high lipophilicity could account for the long duration of action, since fixation to lipids in cutaneous tissues might allow them to act as local depots for slow release of the drug. We have therefore used computer-assisted conformational analysis to investigate the interaction of butenafine with lipids and extended these observations with experimental studies in vitro using liposomes. Conformational analysis of mixed monolayers of phospholipids with the neutral and protonated forms of butenafine highlighted a possible interaction with both the hydrophilic and hydrophobic domains of membrane phospholipids. Studies using liposomes demonstrated that butenafine increases membrane fluidity [assessed by fluorescence polarization of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene and 1,6-diphenylhexatriene] and membrane permeability (studied by release of calcein from liposomes). The results show, therefore, that butenafine readily interacts with lipids and is incorporated into membrane phospholipids. These findings may help explain the excellent antifungal efficacy and long duration of action of this drug when it is used as a topical antifungal agent in humans.

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits dd -carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781–788, 1992), which is rapidly inactivated by many β-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant ( k 2 / K ) for the acylation of the essential serine by benzylpenicillin is 300,000 M −1 s −1 for the Actinomadura sp. strain R39 peptidase, 1,400 M −1 s −1 for B. subtilis PBP4a, and 7,000 M −1 s −1 for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a ( k 2 / K = 46,000 M −1 s −1 ). PBP4a is also much more thermostable than the R39 enzyme.

The cystic fibrosis transmembrane conductance regulator is a cAMP-regulated chloride channel. We used molecular modelling to predict 3-D models for the CFTR membrane domain. Hydropathy and residue conservation in all CFTRs as well as in other proteins suggested that the membrane domain is a 12-helix bundle. If the domain is enclosing a channel for chloride, it could be made of five helices. We propose two structural models in which both lumenal and cytoplasmic entrances to the chloride pore have a ring of positively charged residues. The inner surface of the channel is covered with neutral polar plus one or two charged residues. Helices that are not directly involved in the chloride channel could organise to form a second channel; a dimeric symmetrical structure is proposed. Analysis raised interest for helix 5: this hydrophobic fragment is conserved in all CFTRs and aligns with segments present in several different ion channels and transporters. The existence of an FFXXFFXXF motif is proposed. Helix 5 could be an important domain of CFTRs. The models agree with available data from pathological mutations but does not account for the membrane insertion of a hydrophilic fragment of NBD1.

Dipeptidyl peptidase IV (DPP IV/CD26) belongs to a non-classical subfamily of serine-proteases. Sequence comparisons have identified Asp 599 , Ser 624 , Asp 657 , Asp 702 , and His 734 as highly conserved residues of mouse DPP IV. We previously reported the identification of Ser 624 , Asp 702 and His 734 as the catalytic triad of mouse DPP IV (David, F., Bernard, A. M., Pierres, M., and Marguet, D. (1993) J. Biol. Chem. 268, 17247–17252). Using site-directed mutagenesis, we have shown here that substitution of Asp 599 for Ala (D599A) specifically decreases the cell-surface expression of DPP IV in stably transfected mouse fibroblasts. The D599A mutant remained as a high mannose immature glycoprotein and was rapidly degraded. This retention/degradation process correlates with the generation of a β strand in the C-terminal region of DPP IV as shown by three dimensional computer modeling. Our results suggest that conserved residue Asp 599 is important for the proper folding, glycosylation and transport of mouse DPP IV.

Antibodies are powerful tools for studying the in situ localization and physiology of proteins. The prediction of epitopes by molecular modelling has been used successfully for the papilloma virus, and valuable antibodies have been raised [Muller et al. (1990) J. Gen. Virol., 71, 2709-2717]. We have improved the modelling approach to allow us to predict epitopes from the primary sequences of the cystic fibrosis transmembrane conductance regulator. The procedure involves searching for fragments of primary sequences likely to make amphipathic secondary structures, which are hydrophilic enough to be at the surface of the folded protein and thus accessible to antibodies. Amphipathic helices were predicted using the methods of Berzofsky, Eisenberg and Jahnig. Their hydrophobic-hydrophilic interface was calculated and drawn, and used to predict the orientation of the helices at the surface of the native protein. Amino acids involved in turns were selected using the algorithm of Eisenberg. Tertiary structures were calculated using 'FOLDING', a software developed by R. Brasseur for the prediction of small protein structures [Brasseur (1995) J. Mol. Graphics, in press]. We selected sequences that folded as turns with at least five protruding polar residues. One important property of antibodies is selectivity. To optimize the selectivity of the raised antibodies, each sequence was screened for similarity (FASTA) to the protein sequence from several databanks. Ubiquitous sequences were discarded. This approach led to the identification of 13 potential epitopes in the cystic fibrosis transmembrane conductance regulator: seven helices and six loops.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.

Several shorter analogues of the cell penetrating peptide, transportan, have been synthesized in order to define the regions of the sequence, which are responsible for the membrane translocation property of the peptide. Penetration of the peptides into Bowes melanoma cells and the influence on GTPase activity in Rin m5F cellular membranes have been tested. The experimental data on cell penetration have been compared with molecular modeling of insertion of peptides into biological membranes. Omission of six amino acids from the N-terminus did not significantly impair the cell penetration of the peptide while deletions at the C-terminus or in the middle of the transportan sequence decreased or abolished the cellular uptake. Most transportan analogues exert an inhibitory effect on GTPase activity. Molecular modeling shows that insertion of the transportan analogues into the membrane differs for different peptides. Probably the length of the peptide as well as the location of aromatic and positively charged residues have major impact on the orientation of peptides in the membranes and thereby influence the cellular penetration. In summary, we have designed and characterized several novel short transportan analogues with similar cellular translocation properties to the parent peptide, but with reduced undesired cellular activity.