RESUMEN
The possible influence of thermal motion on 1H chemical shifts is discussed for a small stable protein, the bovine pancreatic Kunitz trypsin inhibitor (BPTI). The thermal effects on the aromatic side chains and on the backbone are treated separately. The thermal motion of the aromatic side chains is accounted for in terms of their rotation around the C(alpha)-C(beta) bond and the motion of each individual proton is interpreted as a ratio between the amount of ordered and quite disordered states. The influence of hydrogen bonds is introduced as an extra contribution to the chemical shifts of the bonded proton. Their contribution to the chemical shifts resulting from the polarization of the peptide bond is investigated, as is their influence on local flexibility. Finally, the relative importance of each contribution to the chemical shift information is compared.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Páncreas/química , Proteínas de Plantas/química , Proteínas/química , Animales , Bovinos , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Químicos , Protones , Temperatura , Inhibidores de Tripsina , alfa-Amilasas/antagonistas & inhibidoresRESUMEN
Essential HTLV-1 biological functions, like host-cell receptor recognition, depend on the structural motives on the surface glycoprotein gp46. We defined a peptide of 88 amino acids [Arg147-Leu234] corresponding to the central part of the protein sequence, where major neutralizing epitopes are localized. After evaluating the feasibility of its chemical synthesis, the chosen sequence was realized using the stepwise solid-phase methodology. Multiple chromatographic purification steps were required to obtain a sample suitable for structural analysis. Correct folding was supported by strong binding of monooclonal antibodies, recognizing known exposed immunodominant regions. Circular dichroism studies confirmed a non-random conformation of at least 70-80% of the synthetic peptide. Investigation of the 3D-structure of the synthetic peptide will provide useful information for future vaccine and drug-design strategies.
Asunto(s)
Productos del Gen env/química , Péptidos/química , Proteínas Oncogénicas de Retroviridae/química , Acetonitrilos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Sitios de Unión/inmunología , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Productos del Gen env/inmunología , Productos del Gen env/metabolismo , Antígenos HTLV-I/química , Antígenos HTLV-I/inmunología , Antígenos HTLV-I/metabolismo , Datos de Secuencia Molecular , Concentración Osmolar , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Proteínas Oncogénicas de Retroviridae/metabolismo , Propiedades de SuperficieRESUMEN
Via the refinement process of the monomer form of an arginine-vasopressin-like insect factor, the paper analyses the most relevant NMR information to define the solution structure of a flexible peptide. The relative importance of the different NOE constraints is discussed.
Asunto(s)
Arginina Vasopresina/química , Saltamontes/química , Oligopéptidos/química , Péptidos Cíclicos/química , Conformación Proteica , Animales , Arginina Vasopresina/análogos & derivados , Arginina Vasopresina/síntesis química , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Oligopéptidos/síntesis química , Péptidos Cíclicos/síntesis química , ProtonesRESUMEN
For flexible peptides, nuclear Overhauser Effects (NOE) experiments do not provide enough information to ensure a correct definition of their solution structure. The use of distance constraints, derived from the knowledge of proton chemical shifts, is developed to restrict the number of possible conformations. In the case of flexible molecules, randomization appears as an important factor of the correct estimation of the chemical shifts from the 3D structure. The refinement of the solution structure of the highly flexible AVP-like parallel dimer is described to illustrate this process.
Asunto(s)
Arginina Vasopresina/química , Espectroscopía de Resonancia Magnética/métodos , Péptidos/química , Protones , Arginina Vasopresina/análogos & derivados , Arginina Vasopresina/síntesis química , Dimerización , Modelos Teóricos , Conformación Proteica , Distribución Aleatoria , SolucionesRESUMEN
The region comprised between the amino acids 175 and 199 of the HTLV-I envelope surface glycoprotein is one of the immunodominant domains of this molecule. In this region, which is well recognized by sera from HTLV-I infected patients, a substitution of the proline at position 192 by a serine has been described in some isolates. Because this mutation could modify the secondary structure of the glycoprotein molecule, we studied the inference of the presence of proline or serine on the recognition of the region 175-199 by human sera. For this, three peptides have been synthetized (a 25-mer 175-199 corresponding to the sequence of the ATK prototype, and two internal 10-mer 190-Pro-199 and 190-Ser-199 having a proline or a serine at position 192) and tested by immunosorbent assay. While most sera reacted with 190-Pro-199 and with 190-Ser-199 synthetic peptides, a differential recognition was observed according to the pathology associated to HTLV-I infection. Moreover sera corresponding to patients infected with a virus harboring a serine at position 192 were found to recognize only the 10-mer with a serine. These data indicates that HTLV-I is subject to antigenic variability.
Asunto(s)
Productos del Gen env/inmunología , Antígenos HTLV-I/inmunología , Infecciones por HTLV-I/inmunología , Proteínas Oncogénicas de Retroviridae/inmunología , Infecciones por HTLV-I/sangre , Humanos , Leucemia de Células T/inmunología , Modelos Moleculares , Fragmentos de Péptidos/inmunologíaRESUMEN
Sequence analysis of the endoglucanase EGCCA of Clostridium cellulolyticum indicates the existence of two domains: a catalytic domain extending from residue 1 to residue 376 and a reiterated domain running from residue 390 to 450. A small deletion in the C terminal end of the catalytic domain inactivated the protein. From the analysis of the sequences of 26 endoglucanases belonging to family A, we focused on seven amino acids which were totally conserved in all the catalytic domains compared. The roles of two of these, Arg-79 and His-122, were studied and defined on the basis of the mutants obtained by introducing various substitutions. Our findings suggest that Arg-79 is involved in the structural organization of the protein; the His-122 residue seems to be more essential for catalysis. The role of His-123, which is conserved only in subfamily A4, was also investigated.
Asunto(s)
Celulasa/genética , Clostridium/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Celulasa/metabolismo , Clostridium/genética , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
The nucleotide sequences of the env genes of seven bovine leukemia viruses and the encoded peptide sequence were compared, with the objective of (i) determining the genetic distance separating bovine leukemia virus isolates from different geographical regions, (ii) identifying particular amino acids that contribute to the sequential and conformational epitopes, and (iii) relating such epitopes to their projected position in a three-dimensional model of the structure of the gp51 surface glycoprotein. Two bovine leukemia virus subgroups were clearly identified, a Japanese-American subgroup represented by strains lambda BLV-1, VdM, and FLK-BLV and a European subgroup by strains T15-2, LB285, and LB59. It was possible to identify amino acids that were important in determining three of the epitopes (F, G, and H) recognized by neutralizing monoclonal and polyclonal antibodies. On the model, these epitopes were adjacent and located on the exposed region of the molecule. Amino acid sequences contributing to a fourth cryptic epitope were identified; as predicted by the model, they lay on the opposite side to the neutralizable epitopes in a region involved in glycoprotein subunit association. The fact that this region is not normally exposed on the virion surface provides further evidence for the validity of the model.
Asunto(s)
Genes Virales , Virus de la Leucemia Bovina/genética , Retroviridae/genética , Proteínas del Envoltorio Viral/genética , Proteínas Estructurales Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , ADN Viral/genética , Epítopos/análisis , Variación Genética , Virus de la Leucemia Bovina/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Mapeo Restrictivo , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
An automatic macromolecular modelling package of unknown protein structures was developed using the intimate correlation which appears between the observed X-ray structures and their associated predicted folding patterns. The method can be considered as a generalization of both the combinatorial [1] and the template identification [2,3] approaches which were proposed some years ago, and provide a fast way of selecting 'structural motifs' to build new proteins. As an illustration, the tertiary fold of the all-beta-domain of the retroviral outermembrane glycoprotein is proposed.
Asunto(s)
Cápside , Glicoproteínas de Membrana , Conformación Proteica , Secuencia de Aminoácidos , Disulfuros , Virus de la Leucemia Murina de Friend , Glicoproteínas , Virus Linfotrópico T Tipo 1 Humano , Virus Linfotrópico T Tipo 2 Humano , Virus de la Leucemia Bovina , Modelos Moleculares , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney , Péptido Hidrolasas , Proteínas de los Retroviridae , TermodinámicaRESUMEN
A new way to predict the topologies of proteins of unknown three-dimensional structure is derived from the comparison of the distribution of the strongest predicted secondary structures with equivalent distributions recorded for proteins of known X-ray structures. As an illustration the tentative three-dimensional model of phosphoribosyl transferases which was proposed by Argos et al. is rediscussed.
Asunto(s)
Pentosiltransferasa , Conformación Proteica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
We proved previously that the distribution of formation energies which may be associated with the predicted secondary structures (or nuclei) is specific of the folding process (Busetta, B. 1986, Biochim. Biophys. Acta 870, 327-338). We developed a new predictive algorithm for protein topologies, based on the search of standard 'folding patterns'. In another manner, the strongest predicted nuclei are used to propose a fast sequence-alignment process which is efficient for distantly related proteins.
Asunto(s)
Algoritmos , Proteínas/clasificación , Alcohol Deshidrogenasa , Secuencia de Aminoácidos , Animales , Cazón , Caballos , Isoenzimas , L-Lactato Deshidrogenasa , Conformación ProteicaRESUMEN
From the prediction of protein secondary structures, formation energies may be estimated for each incipient nucleus of the folding process. The averaging which may be performed on large families of distantly related proteins improves the accuracy of measurement of these energies and the efficiency of the prediction of the different steps involved in the protein folding (secondary structures, domain boundaries, topologies, etc.) and allows the description of new folding patterns.
Asunto(s)
Conformación Proteica , Proteínas , Secuencia de Aminoácidos , Matemática , Relación Estructura-Actividad , TermodinámicaRESUMEN
The ab initio prediction of the domain boundaries and types remains one of the most important problems which makes a possible description of protein tertiary structure from its amino-acid sequence elusive. The present paper describes new methods to predict both of them. The predictions of domain boundaries and types in proteins of known X-ray structures are reported in order to check the efficiency of these new algorithms.
Asunto(s)
Conformación Proteica , Proteínas , Secuencia de Aminoácidos , Animales , Humanos , Sustancias Macromoleculares , Matemática , Modelos Estructurales , Difracción de Rayos XRESUMEN
The estimates of the different contributions to the free energy of folded proteins are derived from analysis of protein X-ray structures, and introduced in the conformational analysis of the protein tertiary structures at a very macroscopic level. Different predictions of protein topologies are reported in the case of all-alpha and all-beta proteins.