RESUMEN
We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.
Asunto(s)
Páncreas/enzimología , Elastasa Pancreática/metabolismo , Anilidas/metabolismo , Animales , Sitios de Unión , Cromatografía , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Cinética , Masculino , Peso Molecular , Elastasa Pancreática/antagonistas & inhibidores , Elastasa Pancreática/aislamiento & purificación , Péptidos/metabolismo , Péptidos/farmacología , Ratas , Ratas Endogámicas , Homología de Secuencia de Ácido Nucleico , Porcinos , Ácido TrifluoroacéticoRESUMEN
Nowadays, the use of synthetic carriers as biochemical reagents and immunogens is entering a new phase. The multimeric nature of these constructs, the unambiguous composition and the ease, reliability and versatility of their production, make this type of carriers well-suited to various biotechnological and biochemical applications for diagnostic purposes, protein mimetics, antiviral agents, vaccines, drug and gene delivery vehicles. This review aims to briefly summarize the different types of synthetic carriers currently in use and will be focused on an innovative type of multifunctional helicoid carrier-foldamer, named SOC(n)-I, II, which was successfully developed in our laboratory. Our concept was to construct an artificial template with structural rigidity and regularity, so as the peptide epitopes/pharmacophore groups could be anchored without any conformational restriction and steric hindrance as demonstrated by conformational studies using (1)H-NMR, CD and FT-IR. SOC(n)-I,II were used as antigenic substrates in developing immunoassays of high sensitivity, specificity and reproducibility, as well as potent immunogens to generate site-specific antibodies. To this end, we emphasize on the application of SOC(n) carriers covalently bearing a 'built-in' adjuvant, for the preparation of totally synthetic peptide-based vaccines for human use.
Asunto(s)
Portadores de Fármacos/síntesis química , Oligopéptidos/síntesis química , Vacunas Sintéticas/inmunología , Secuencia de Aminoácidos , Dicroismo Circular , Epítopos/química , Epítopos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Oligopéptidos/inmunología , Polímeros/síntesis química , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The Arg-Gly-Asp RGD motif of adhesive proteins is recognized by the activated platelet integrin alpha(IIb)beta3. Binding of fibrinogen (Fg) to activated alpha(IIb)beta3 causes platelet aggregation and thrombus formation. Highly constraint cyclic (S,S) -CXaaC- containing peptides incorporating the (S,S) -CDC- and (S,S) -CRC- motifs were tested for their ability to inhibit platelet aggregation and Fg binding. Our results suggest that the above cyclic scaffolds stabilize a favorable structure for the antiaggregatory activity (IC50-values ranged from 1.7 to 570 microm). The peptides inhibited Fg binding with IC50-values up to 30-fold lower than those determined for the inhibition of the adenosine diphosphate (ADP)-induced platelet aggregation. Importantly, peptides (S,S) PSRCDCR-NH2 (peptide 11) and (S,S) PRCDCK-NH2 (peptide 10) did not inhibit PAC-1 binding to the activated platelets at a concentration in which they completely inhibited Fg binding. Moreover, (S,S) PSRCDCR-NH(2) (peptide 11), one of the more active peptides, inhibited ADP-induced P-selectin exposure. By contrast, peptide (S,S) Ac-RWDCRC-NH2, incorporating the inverse (S,S) -DCRC- sequence (peptide 16), failed to inhibit P-selectin exposure whereas at the same concentration, it effectively inhibited PAC-1 and Fg binding. It is concluded that peptides containing the (S,S) -CDC- as well the (S,S) -CRC- sequences, exhibit a broad range of activities toward platelets, and could be helpful tools for elucidating the structural interaction of Fg with the integrin receptor alpha(IIb)beta3, in its activated form. Furthermore, the (S,S) -RCDC- sequence can be used as a scaffold for developing potent non-RGD-like Fg-binding inhibitors.
Asunto(s)
Plaquetas/metabolismo , Fibrinógeno/antagonistas & inhibidores , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Fosfatasa 2 de Especificidad Dual , Fibrinógeno/metabolismo , Humanos , Concentración 50 Inhibidora , Conformación Molecular , Selectina-P , Péptidos Cíclicos/síntesis química , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/química , Unión Proteica/efectos de los fármacos , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/metabolismo , Relación Estructura-ActividadRESUMEN
Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Epítopos/inmunología , Miastenia Gravis/inmunología , Receptores Nicotínicos/inmunología , Adulto , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Enfermedades Autoinmunes/epidemiología , Enfermedades Autoinmunes/terapia , Bungarotoxinas/farmacología , Modelos Animales de Enfermedad , Epítopos/ultraestructura , Femenino , Humanos , Incidencia , Recién Nacido , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Miastenia Gravis/epidemiología , Miastenia Gravis/terapia , Fragmentos de Péptidos/inmunología , Embarazo , Conformación Proteica , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Torpedo/genéticaRESUMEN
The ionization state of Leu-enkephalin in DMSO and MeCN/DMSO (4/1) solution was studied by the combined use of 17O NMR and FT-IR spectroscopy. After lyophilization of an aqueous solution at nearly neutral pH, Leu-enkephalin essentially exists in the uncharged state in MeCN/DMSO (4/1) solution. In pure DMSO, only 40% of the Leu-enkephalin molecules are in the zwitterionic state under the same conditions.
Asunto(s)
Encefalina Leucina/química , Péptidos/química , Solventes , Secuencia de Aminoácidos , Dimetilsulfóxido , Análisis de Fourier , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
A sensitive, highly reproducible, solid-phase enzyme immunoassay (ELISA), was developed in order to investigate whether the synthetic heptapeptide PPGMRPP-a major epitope of the Sm autoantigen-anchored in five copies to a sequential oligopeptide carrier (SOC), [(PPGMRPP)5-SOC5] is a suitable antigenic substrate to identify anti-Sm/antibodies. Sera with different autoantibody specificities [45 anti-Sm, 40 anti-U1RNP, 40 anti-Ro (SSA)/La(SSB) positive, 21 Antinuclear antibody positive, but negative for antibodies to extractable nuclear antigens (ANA + /ENA - ) and 75 normal human sera, ANA negative] and 75 sera from patients with rheumatoid arthritis (RA) were tested for anti-(PPGMRPP)5-(SOC)5 reactivity in order to evaluate the specificity and sensitivity of the method to detect anti-Sm antibodies. RNA immunoprecipitation assays for the detection of anti-Sm and anti-U1RNP antibodies and counter immunoelectrophoresis (CIE) for the detection of anti-Ro(SSA) and anti-La(SSB) antibodies were used as reference techniques. The sensitivity of the method was 98% and the specificity was 68% for the determination of anti-Sm antibodies, while for the determination of anti-Sm and/or anti-U1RNP reactivity (antibodies to snRNPs) the corresponding values were 82% and 86%, respectively. In a comparison of the above assay with an ELISA, using Sm/U1RNP purified complex as immobilized antigen it was shown that the sensitivity of the anti-Sm/U1RNP ELISA in detecting anti-snRNPs was 74%; in addition sera with anti-Sm antibodies gave higher binding in the anti-(PPGMRPP)5-(SOC)5 ELISA compared with anti-Sm/U1RNP ELISA. Intra- and inter-assay precision was measured on four sera with reactivities extending into a wide range of absorbance values showed that the intra-assay coefficient of variation (CV%) ranged from 2.7 to 6 and the inter-assay CV% ranged from 9 to 14.5. These results indicate that the PPGMRPP peptide anchored to a pentameric SOC as a carrier is a suitable antigen for detecting anti-Sm antibodies and that the above ELISA is a rapid, reproducible and valuable screening method to test anti-Sm/U1RNP reactivities.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Autoantígenos/inmunología , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Oligopéptidos , Ribonucleoproteínas Nucleares Pequeñas , Anticuerpos Antinucleares/aislamiento & purificación , Unión Competitiva , Portadores de Fármacos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas Nucleares snRNPRESUMEN
OBJECTIVE: Thyroglobulin (Tg) is a large autoantigen involved in autoimmune thyroid diseases. Tg epitopes have, so far, been identified within large peptides. In the present study, we used small synthetic peptides to finely map serological epitopes on the highly immunogenic C-terminal region of Tg. Homology of this region to acetylcholinesterase (AChE) has been implicated in the pathogenesis of thyroid eye disease (TED) through cross-reactive antibodies. METHODS: We tested total IgG purified from four pilot Graves' disease (GD) sera reactive with both Tg and AChE and from three healthy controls, for reactivity against overlapping 20mer peptides (pin synthesis) covering the sequence 2171-2748 of human Tg. Antibody-reactive peptides were subsequently synthesized by a solid-phase technique for confirmation with a large number of sera: 99 GD, 32 Hashimoto's thyroiditis (HT) and 45 healthy controls. RESULTS: Peptides TgP15, TgP26 and TgP41 (amino acids 2339-2358, 2471-2490 and 2651-2670 respectively) were found to be targets of autoantibodies on intact Tg, recognized by a statistically significant proportion of GD sera (22.2%, 35.4% and 30.3% respectively), compared with HT (0%, 15.6% and 6.3% respectively) and healthy controls (0%, 4.4% and 4.4% respectively). The majority of GD sera (56.6%) were positive for at least one of the three peptides. In GD, TgP26 reactivity was found to be associated with TED (48.6% with TED versus 25.5% without TED, P<0.05). CONCLUSION: Some epitopes on the C-terminal region of Tg are associated with GD. A subset of Tg-reactive autoantibodies, directed to this region, is associated with TED and may be involved in the development of the disease.
Asunto(s)
Acetilcolinesterasa/inmunología , Autoantígenos/inmunología , Epítopos de Linfocito B/inmunología , Enfermedad de Graves/inmunología , Tiroglobulina/inmunología , Tiroiditis Autoinmune/inmunología , Acetilcolinesterasa/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Autoanticuerpos/inmunología , Inhibidores de la Colinesterasa/inmunología , Ensayo de Inmunoadsorción Enzimática , Enzimas Inmovilizadas/inmunología , Enzimas Inmovilizadas/metabolismo , Mapeo Epitopo , Femenino , Enfermedad de Graves/enzimología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Tiroiditis Autoinmune/enzimologíaRESUMEN
Auto-antibodies to the nicotine acetylcholine receptor (AChR) cause the disease myasthenia gravis (MG). Animals immunized with AChR or receiving anti-AChR antibodies acquire MG symptoms. The majority of the monoclonal antibodies (mAbs) raised in rats against intact AChR bind to a region on the extracellular side of the AChR's alpha-subunit, the main immunogenic region (MIR). The major loop of the overlapping epitopes for several anti-MIR mAbs has been localised between residues 67-76 of the alpha-subunit. Anti-MIR mAbs are very potent in accelerating AChR degradation (antigenic modulation) in muscle cell cultures and transferring experimental MG in animals. Fab fragments of single anti-MIR mAbs when bound to the AChR inhibit two-thirds of the MG patients' antibodies from binding and from inducing antigenic modulation of the AChR. This suggest that the majority of the human MG antibodies are also directed against the MIR. It has however to be verified by direct experiments.
Asunto(s)
Miastenia Gravis/inmunología , Receptores Nicotínicos/inmunología , Animales , Anticuerpos Monoclonales , Autoinmunidad , Reacciones Cruzadas , Epítopos/inmunología , Humanos , Conformación MolecularRESUMEN
The Arg-Leu-Gly tripeptide is the repeating fragment of sequential arginine-rich polypeptides capable of interacting with DNA. The conformational influence of solvent molecules (DMSO/H2O) were investigated with the combined use of molecular dynamics and energy minimization. It was found that water molecules greatly contribute to the peptide structure by solvating all its hydrophylic sites even in the presence of DMSO excess, whereas one water molecule links the ammonium and carboxylic ends of the Arg-Leu-Gly. The persistence of residual water, which was confirmed by varying the computer simulation parameters, indicates that pretreatment of peptide segments in aqueous solutions should greatly affect their conformational properties in organic media. A satisfactory agreement between experimental data (1H-NMR and IR spectroscopy) and the presented computational study deserves also to be noted.
Asunto(s)
Dimetilsulfóxido/química , Oligopéptidos/química , Péptidos/química , Agua/química , Secuencia de Aminoácidos , Análisis por Conglomerados , Gráficos por Computador , Simulación por Computador , Espectroscopía de Resonancia Magnética , Modelos Químicos , Datos de Secuencia Molecular , Conformación Proteica , Solventes/químicaRESUMEN
A series of biologically active peptides and their conjugates with lipidic amino acids were investigated by systematic change of the mobile phase composition using traditional octadecylsilica stationary phase and the newly developed Supelcosil LC-ABZ column. The mobile phases contained various concentrations of methanol and acetonitrile combined with 0.1% trifluoroacetic acid (TFA). Better peak shapes and higher resolution of the isomers could be observed when the mobile phase contained 0.1% TFA. More symmetrical peaks and much higher S values (slope of the log k' vs organic phase concentration plots) were obtained on the special reversed-phase column developed for anionic, basic or zwitterionic compounds. The optimum separation conditions were scaled up to a semi-preparative reversed-phase column (15 mm i.d.) to collect mg quantities of isomers for further studies.
Asunto(s)
Aminoácidos/análisis , Péptidos/análisis , Acetonitrilos , Secuencia de Aminoácidos , Aminoácidos/química , Cromatografía Líquida de Alta Presión/métodos , Datos de Secuencia Molecular , Péptidos/química , Estereoisomerismo , Relación Estructura-Actividad , Ácido TrifluoroacéticoRESUMEN
Interactions of calf thymus DNA with sequential polypeptides were studied using c.d. spectroscopy in aqueous solutions. It was found that DNA structural alterations induced by sequential polypeptides (L-Arg-X-Gly)n (where X = L-Val, Leu, Ile, Nva, Nle) are modulated by the nature of the X residue. Thus, the polypeptide (L-Arg-L-Nva-Gly)n induced the 10.2B-DNA form, whereas the polypeptides (L-Arg-L-Ile-Gly)n having one methyl group less on the X residue side chain, did not provide any significant modification to the structure of DNA. The effect of ionic strength from 0.14 M NaCl (physiological value) to zero was also analysed on the basis of the observed c.d. changes and the degree of complexation in the DNA-polypeptides was estimated.
Asunto(s)
ADN/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Bovinos , Dicroismo Circular , Electroquímica , Datos de Secuencia Molecular , Soluciones , Timo/metabolismoRESUMEN
Interactions between calf thymus DNA and (L-Arg-X-Gly)n sequential polypeptides (where X = L-Ala, Val, Leu, Ile, Nva, Nle) in trifluoroethanol: water (40:60) solutions in the salt range of 0.12-0.5 M NaCl, were studied using c.d. spectroscopy. It was found that DNA tertiary structure (psi form) is modulated by the nature of the polypeptides (variation of X residue). The effect of the secondary structure of polypeptides on the formation of psi-DNA was also analysed. Unordered polypeptides destabilized psi aggregates, while helical polypeptides favoured DNA tertiary structure. A loss of tertiary structure was observed in the presence of the (L-Arg-L-Val-Gly)n, which can be attributed to the ability of valine to suppress psi-type DNA.
Asunto(s)
ADN/química , Péptidos/química , Trifluoroetanol/química , Secuencia de Aminoácidos , Animales , Bovinos , Dicroismo Circular , Datos de Secuencia Molecular , Soluciones , Timo/metabolismoRESUMEN
A new class of sequential oligopeptide carriers (SOCn), namely (Lys-Aib-Gly)n (n = 2-7), for anchoring antigenic peptides, is presented. These SOCn have been designed in order to assume a determined structural motif, exhibiting defined spatial orientations of the Lys-N epsilon H2 anchoring groups. The NMR study showed that SOCn adopt a rigid conformation with some regularity, initiated from the C-terminus of the carrier, while molecular dynamics simulation confirmed the occurrence of a distorted 3(10)-helix. It was also demonstrated, by 1HNMR, that all the antigenic peptides bound to the SOCn retain their original, folded active, structure and that probably they do not interact to each other. It is concluded that the beneficial structural elements of the SOCn impose a favorable disposition of the anchored peptides so that potent antigens with maximum molecular recognition are generated.
Asunto(s)
Oligopéptidos/química , Oligopéptidos/inmunología , Receptores Colinérgicos/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Epítopos/química , Epítopos/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Oligopéptidos/síntesis química , Conformación Proteica , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismoRESUMEN
The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti-MIR monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67-76 of the AChR alpha-subunit. A thorough study of the epitope(s) for anti-MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data. Use of all possible overlapping hexapeptides within Torpedo and human alpha 40-91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide alpha 67-71. Systematic screening of peptide analogues, where each amino acid residue within alpha 67-76 and alpha 67-74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti-MIR mAb binding, whereas Pro69 and Ala/Asp 70 were less but still significantly important. mAb binding to alpha 67-76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of alpha 76 or combinations of two substitutions at alpha 73 and alpha 76 were tested. Several of these analogues (mainly His76, Arg76, Val73Ala76, His73Ala76, Val73Arg76) exhibited dramatic mAb binding enhancement. Some anti-MIR mAbs that do not bind to alpha 67-76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.
Asunto(s)
Receptores Nicotínicos/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Electrophorus , Epítopos , Humanos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Relación Estructura-Actividad , TorpedoRESUMEN
The sequential polypeptides (L-Arg-Xaa-Gly)n where Xaa represents amino acid residues Ala, Val and Leu, were employed as models of arginine-rich histones, in studying their interactions with nucleic acids. These polypeptide-DNA complexes were prepared using gradient dialysis and their conformational properties were investigated by circular dichroism spectroscopy. It was found that poly(L-Arg-L-Val-Gly) caused pronounced structural changes in the DNA molecule (conformational transition from B to the more compact and asymmetric C form) as a function of ionic strength and polypeptide: DNA ratio. In contrast the DNA interaction with poly (L-Arg-L-Ala-Gly) and poly (L-Arg-L-Leu-Gly) increased in the order of Ala----Leu, but with slight structural changes in the DNA secondary conformation. Thus, the importance of the composition, amino acid sequence and conformation of the polypeptides which bind to DNA was demonstrated. The significance of the hydrophobic forces besides the arginine-phosphate charge interaction, which modulate the nature of the polypeptide-DNA complexes and their condensation into higher-ordered tertiary structures, as found in chromatin, was also confirmed.
Asunto(s)
Cromatina/metabolismo , ADN/metabolismo , Sustancias Macromoleculares/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/análisis , Secuencia de Bases , Bovinos , Dicroismo Circular , Histonas/análisis , Modelos Químicos , Unión Proteica , Conformación Proteica , Espectrofotometría Ultravioleta , Relación Estructura-ActividadRESUMEN
The three retro-analogs of the tBuCO-Ala-Gly-NHiPr dipeptide, in which each amide bond had been successively reversed, were studied in solution by 1H-n.m.r. and i.r. spectroscopy with reference to the conformational properties of their parent dipeptide. Reversal of the Ala-Gly amide bond proved to perturb the folding tendency of the backbone less than the inversion of either of the terminal amide bonds. The crystal structure of the retro-peptide containing a reversed Ala-Gly amide bond was also solved by X-ray diffraction and constitutes the first available data for this retro-peptide series. In contrast to the beta II-folded structure of the parent dipeptide, the retro-peptide molecule adopts an open conformation in the crystal.
Asunto(s)
Dipéptidos , Péptidos Cíclicos , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Soluciones , Espectrofotometría Infrarroja , Difracción de Rayos XRESUMEN
The synthesis of Leu-enkephalin selectively 17O-enriched in Gly2 and Gly3 is reported. The 17O-nmr chemical shifts of [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in H2O are almost identical and independent of the pH. Since hydrogen bonding is the dominant factor governing the chemical shifts of the peptide oxygen, it can be concluded that the hydration state of both oxygens is identical and independent of the pH. The 17O chemical shifts of the [17O-Leu5]-enkephalin terminal carboxyl group at pH approximately 1.9 and 5.6 are very different in H2O but very similar in CH3CN/DMSO (4:1) solution. This suggests that the protonation state of the carboxyl group at both pH values in CH3CN/DMSO solution is the same and consequently that Leu-enkephalin exists in the neutral form at pH approximately 5.6. In this organic mixed solvent system both Gly2 and Gly3 oxygen resonances exhibit a significant shift to high frequency by the same extent (delta delta approximately 30 ppm). It is concluded that both peptide oxygens are not hydrogen bonded to an appreciable extent and that no specific 2----5 hydrogen bonding exists to an appreciable extent. This conclusion is in agreement with the energy of activation for molecular rotation, as determined from T1 measurements, which was found to be almost identical for both [17O-Gly2, Leu5]- and [17O-Gly3, Leu5]-enkephalins in CH3CN/DMSO (4:1) mixed solvent.
Asunto(s)
Encefalinas , Espectroscopía de Resonancia Magnética/métodos , Isótopos de Oxígeno , Conformación Proteica , SolucionesRESUMEN
Leucine-enkephalin (Try1-Gly2-Gly3-Phe4-Leu5) has been crystallized as a trihydrate from water solution. X-ray diffraction reveals a tightly folded molecular conformation with two fused beta III- (Gly2-Gly3) and beta I- (Gly3-Phe4) turns. The Tyr1 and Phe4 aromatic rings have a close orthogonal arrangement analogous to the tyramine and cyclohexenyl rings in morphine. This suggests that the conformation found in the trihydrate crystal structure could be required for recognition by mu-receptor sites.
Asunto(s)
Encefalina Leucina , Morfina , Modelos Moleculares , Conformación Molecular , Conformación Proteica , Difracción de Rayos XRESUMEN
Autoantibodies in sera from patients with systemic lupus erythematosus (SLE) and onchocerciasis recognize calreticulin (CaR), a calcium-binding protein, as antigen. In this study we present the immunological properties of two synthetic peptides prepared to correspond to the 1-24 and 7-24 amino acid sequence of CaR. In contrast to information previously reported for the recombinant protein, the CaR-peptide analogues appeared immunoreactive to anti-Ro/SSA autoimmune sera. Human sera from patients with SLE, Sjögren's syndrome (SS), rheumatoid arthritis (RA), as well as mixed connective tissue disease (MCTD), demonstrated a positive autoimmune response (binding of antibodies), to the CaR-peptide analogues. These findings suggest that anti-calreticulin autoantibodies are not restricted to any disease specificity.
Asunto(s)
Autoanticuerpos/inmunología , Proteínas de Unión al Calcio/inmunología , Enfermedades del Tejido Conjuntivo/inmunología , Lupus Eritematoso Sistémico/inmunología , Péptidos/inmunología , ARN Citoplasmático Pequeño , Enfermedades Reumáticas/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Autoantígenos , Calreticulina , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Immunoblotting , Inmunoelectroforesis , Datos de Secuencia Molecular , Péptidos/síntesis química , RibonucleoproteínasRESUMEN
The peptide trifluoroacetyl-Leu-Ala-(p-trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 x 10(-8) M. Co-crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 A effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 A and 2 degrees, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe-215 in a geometry that resembles the previously reported structure of the CF3-Lys-Ala complex at 2.5 A, in a parallel beta-sheet association with the loop 214-216. The analysis of the close contacts (less than 3.5 A) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu-Ala or Ala-anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191-195 and 213-215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.