RESUMO
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.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Pâncreas/química , Proteínas de Plantas/química , Proteínas/química , Animais , Bovinos , Cristalografia por Raios X , Ligação de Hidrogênio , Modelos Químicos , Prótons , Temperatura , Inibidores da Tripsina , alfa-Amilases/antagonistas & inibidoresRESUMO
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.
Assuntos
Produtos do Gene env/química , Peptídeos/química , Proteínas Oncogênicas de Retroviridae/química , Acetonitrilas/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Sítios de Ligação/imunologia , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Produtos do Gene env/imunologia , Produtos do Gene env/metabolismo , Antígenos HTLV-I/química , Antígenos HTLV-I/imunologia , Antígenos HTLV-I/metabolismo , Dados de Sequência Molecular , Concentração Osmolar , Peptídeos/imunologia , Peptídeos/metabolismo , Ligação Proteica/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Proteínas Oncogênicas de Retroviridae/metabolismo , Propriedades de SuperfícieRESUMO
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.
Assuntos
Arginina Vasopressina/química , Gafanhotos/química , Oligopeptídeos/química , Peptídeos Cíclicos/química , Conformação Proteica , Animais , Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/síntese química , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/síntese química , Peptídeos Cíclicos/síntese química , PrótonsRESUMO
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.
Assuntos
Arginina Vasopressina/química , Espectroscopia de Ressonância Magnética/métodos , Peptídeos/química , Prótons , Arginina Vasopressina/análogos & derivados , Arginina Vasopressina/síntese química , Dimerização , Modelos Teóricos , Conformação Proteica , Distribuição Aleatória , SoluçõesRESUMO
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.
Assuntos
Produtos do Gene env/imunologia , Antígenos HTLV-I/imunologia , Infecções por HTLV-I/imunologia , Proteínas Oncogênicas de Retroviridae/imunologia , Infecções por HTLV-I/sangue , Humanos , Leucemia de Células T/imunologia , Modelos Moleculares , Fragmentos de Peptídeos/imunologiaRESUMO
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.
Assuntos
Celulase/genética , Clostridium/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Celulase/metabolismo , Clostridium/genética , Análise Mutacional de DNA , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
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.
Assuntos
Genes Virais , Vírus da Leucemia Bovina/genética , Retroviridae/genética , Proteínas do Envelope Viral/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Viral/genética , Epitopos/análise , Variação Genética , Vírus da Leucemia Bovina/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Mapeamento por Restrição , Proteínas do Envelope Viral/imunologiaRESUMO
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.
Assuntos
Capsídeo , Glicoproteínas de Membrana , Conformação Proteica , Sequência de Aminoácidos , Dissulfetos , Vírus da Leucemia Murina de Friend , Glicoproteínas , Vírus Linfotrópico T Tipo 1 Humano , Vírus Linfotrópico T Tipo 2 Humano , Vírus da Leucemia Bovina , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney , Peptídeo Hidrolases , Proteínas dos Retroviridae , TermodinâmicaRESUMO
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.
Assuntos
Pentosiltransferases , Conformação Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , Relação Estrutura-Atividade , Difração de Raios XRESUMO
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.
Assuntos
Algoritmos , Proteínas/classificação , Álcool Desidrogenase , Sequência de Aminoácidos , Animais , Cação (Peixe) , Cavalos , Isoenzimas , L-Lactato Desidrogenase , Conformação ProteicaRESUMO
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.
Assuntos
Conformação Proteica , Proteínas , Sequência de Aminoácidos , Matemática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
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.
Assuntos
Conformação Proteica , Proteínas , Sequência de Aminoácidos , Animais , Humanos , Substâncias Macromoleculares , Matemática , Modelos Estruturais , Difração de Raios XRESUMO
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.