RESUMEN
The recently described site of Kalinga in the Philippines adds to our understanding of Early-Middle Pleistocene hominin behaviour. Yet, disentangling the natural from the anthropogenic modifications that have taken place in such an old archaeological site is challenging. In this paper we use a set of taphonomic tools at hand to rectify the distortion made by natural processes during the formation of the Kalinga site. From the description of the ribs completeness, surface damages and scattering in the excavation, one can reconstruct the butchery, transport and deposition sequence of the rhino carcass and its post-depositional disturbances and diagenetic evolution of the site. We conclude that the rhino and the stone artefacts potentially used to deflesh the carcass were transported by a mudflow from its butchery place over a few meters only and got stuck and mixed with an older faunal assemblage that was transported by a small stream.
RESUMEN
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.
Asunto(s)
Membrana Celular/química , Fosfolípidos/química , Proteínas Recombinantes de Fusión/química , Alcoholes , Secuencia de Aminoácidos , Permeabilidad de la Membrana Celular , Diseño de Fármacos , GTP Fosfohidrolasas/química , Galanina , Humanos , Radioisótopos de Yodo , Membrana Dobles de Lípidos/química , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Células Tumorales Cultivadas , Venenos de Avispas , AguaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Biología Computacional/métodos , Hexosiltransferasas , Peptidil Transferasas , Proteínas/química , Proteínas/metabolismo , Algoritmos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Apolipoproteínas E/química , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Arginina/análisis , Arginina/metabolismo , Sitios de Unión , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Bases de Datos Factuales , Diseño de Fármacos , Productos del Gen gag/química , Productos del Gen gag/genética , Productos del Gen gag/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Mutación/genética , Proteínas de Unión a las Penicilinas , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/antagonistas & inhibidores , Proteínas/genética , Sensibilidad y Especificidad , Factores de Tiempo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
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.
Asunto(s)
Defensinas , Hormonas de Insectos/química , Espectroscopía de Resonancia Magnética , Péptidos Cíclicos , Péptidos/química , Proteínas de Transferencia de Fosfolípidos , Proteínas/química , Secuencia de Aminoácidos , Antiinfecciosos/química , Proteínas Bacterianas/química , Proteínas Portadoras/química , Lipopéptidos , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Proteínas de Plantas/química , Conformación Proteica , Soluciones , Relación Estructura-Actividad , Tensoactivos/químicaRESUMEN
Among different ab initio approaches to calculate 3D-structures of proteins out of primary sequences, a few are using restricted dihedral spaces and empirical equations of energy as is OSIRIS. All those approaches were calibrated on a few proteins or fragments of proteins. To optimize the calculation over a larger diversity of structures, we need first to define for each sequence what are good conditions of calculations in order to choose a consensus procedure fitting most 3D-structures best. This requires objective classification of calculated 3D-structures. In this work, populations of avian and bovine pancreatic polypeptides (APP, BPP) and of calcium-binding protein (CaBP) are obtained by varying the rate of the angular dynamics of the second step of OSIRIS. Then, 3D-structures are clustered using a nonhierarchical method, SICLA, using rmsd as a distance parameter. A good clustering was obtained for four subpopulations of APP, BPP and CaBP. Each subpopulation was characterized by its barycenter, relative frequency and dispersion. For the three alpha-helix proteins, after the step 1 of OSIRIS, most secondary structures were correct but molecules have a few atomic contacts. Step 2, i.e., the angular dynamics, resolves those atomic contacts and clustering demonstrates that it generates subpopulations of topological conformers as the barycenter topologies show.
Asunto(s)
Modelos Moleculares , Proteínas/química , Programas Informáticos , Algoritmos , Secuencia de Aminoácidos , Animales , Aves , Proteínas de Unión al Calcio/química , Bovinos , Datos de Secuencia Molecular , Polipéptido Pancreático/química , Conformación ProteicaRESUMEN
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 NBDI.
Asunto(s)
Simulación por Computador , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Secuencia Conservada , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1-H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site.
Asunto(s)
Naftalenosulfonatos de Anilina , Colorantes Fluorescentes , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Ionóforos/farmacología , Potasio/metabolismo , Pliegue de Proteína , Valinomicina/farmacología , Adenosina Difosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Modelos Moleculares , Potasio/farmacología , Estómago/enzimologíaRESUMEN
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 [Müller 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 Jähnig. 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.
Asunto(s)
Antígenos de Superficie/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Epítopos/química , Estructura Secundaria de Proteína , Proteínas/química , Algoritmos , Secuencia de Aminoácidos , Antígenos de Superficie/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Análisis de Fourier , Humanos , Microcomputadores , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Valor Predictivo de las Pruebas , Proteínas/inmunología , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
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 beta-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.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/aislamiento & purificación , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Peptidil Transferasas , Bacillus subtilis/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Cinética , Lactamas/metabolismo , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/genética , Proteínas de Unión a las Penicilinas , Plásmidos , Tioléster Hidrolasas/metabolismoRESUMEN
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.
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Antifúngicos/química , Bencilaminas/química , Lípidos/química , Naftalenos/química , Fluoresceínas/química , Polarización de Fluorescencia , Liposomas , Fluidez de la Membrana , Membranas Artificiales , Conformación Molecular , Permeabilidad , Fosfolípidos/química , EstereoisomerismoRESUMEN
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.
Asunto(s)
Proteínas Portadoras/síntesis química , Portadores de Fármacos , Péptidos/síntesis química , Péptidos/farmacocinética , Proteínas Recombinantes de Fusión/síntesis química , Secuencia de Aminoácidos , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Péptidos de Penetración Celular , Galanina , Humanos , Melanoma , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Conformación Proteica , Proteínas Recombinantes de Fusión/farmacocinética , Células Tumorales Cultivadas , Venenos de AvispasRESUMEN
Dipeptidyl peptidase IV (DPP IV/CD26) belongs to a non-classical subfamily of serine-proteases. Sequence comparisons have identified Asp599, Ser624, Asp657, Asp702, and His734 as highly conserved residues of mouse DPP IV. We previously reported the identification of Ser624, Asp702 and His734 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 Asp599 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 beta strand in the C-terminal region of DPP IV as shown by three dimensional computer modeling. Our results suggest that conserved residue Asp599 is important for the proper folding, glycosylation and transport of mouse DPP IV.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico/química , Compartimento Celular , Membrana Celular/enzimología , Dipeptidil Peptidasa 4/ultraestructura , Glicosilación , Ratones , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Epítopos/inmunología , Mucosa Gástrica/enzimología , ATPasa Intercambiadora de Hidrógeno-Potásio/inmunología , Modelos Moleculares , Adenosina Trifosfato/metabolismo , Algoritmos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Unión Competitiva , Western Blotting , Reacciones Cruzadas , Epítopos/química , Epítopos/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , ATPasa Intercambiadora de Hidrógeno-Potásio/química , ATPasa Intercambiadora de Hidrógeno-Potásio/metabolismo , Técnicas de Inmunoadsorción , Microsomas/enzimología , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Desnaturalización Proteica , Estructura Secundaria de Proteína , Inhibidores de la Bomba de Protones , Conejos , Porcinos , TermodinámicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras , Proteínas de Escherichia coli , Escherichia coli/química , Hexosiltransferasas/fisiología , Complejos Multienzimáticos/fisiología , Muramoilpentapéptido Carboxipeptidasa , Peptidoglicano Glicosiltransferasa , Peptidil Transferasas/fisiología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Hexosiltransferasas/química , Hexosiltransferasas/genética , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Proteínas de Unión a las Penicilinas , Péptidos/química , Péptidos/genética , Péptidos/fisiología , Peptidil Transferasas/química , Peptidil Transferasas/genética , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Defensinas , Hormonas de Insectos/química , Secuencia de Aminoácidos , Animales , Hidrógeno , Insectos , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Recombinantes/química , Saccharomyces cerevisiae/química , TermodinámicaRESUMEN
Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins.