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1.
J Mol Biol ; 290(3): 717-30, 1999 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-10395825

RESUMO

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , GTP Fosfo-Hidrolases/química , Proteínas de Membrana/química , Proteínas/genética , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Dinaminas , Proteína Adaptadora GRB2 , GTP Fosfo-Hidrolases/metabolismo , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Proteínas Son Of Sevenless , Espectrometria de Fluorescência , Ressonância de Plasmônio de Superfície
2.
Bioorg Med Chem Lett ; 8(11): 1419-24, 1998 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-9871777

RESUMO

A new series of 4-substituted pipecolic acid derivatives was prepared and incorporated into dipeptoids. The resulting products behave as moderately potent CCK-B antagonists but their constrained structure and its comparison with structurally related compounds yield valuable information about the conformational requirements for optimal recognition of the CCK-B receptor by antagonists.


Assuntos
Colecistocinina/metabolismo , Ácidos Pipecólicos/síntese química , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Células CHO , Córtex Cerebral/metabolismo , Cricetinae , Cobaias , Técnicas In Vitro , Modelos Moleculares , Conformação Molecular , Pâncreas/metabolismo , Ácidos Pipecólicos/química , Ácidos Pipecólicos/farmacologia , Conformação Proteica , Ratos , Receptor de Colecistocinina B , Relação Estrutura-Atividade
3.
Eur J Biochem ; 226(2): 413-22, 1994 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-8001559

RESUMO

Two-dimensional and three-dimensional 1H-NMR experimental data [Simorre, J. P., Caille, A., Marion, D., Marion, D. & Ptak, M. (1991) Biochemistry 30, 11600-11608] were used to build models of the three-dimensional structure of a non-specific wheat lipid-transfer protein (LTP) by using distance geometry, simulated annealing, energy minimization and molecular dynamics techniques. A first set of 881 distance constraints derived from NOE cross-peak intensities was used to generate 74 initial structures. One family of topological mirror images of the protein structure was eliminated by considering helical secondary-structure organization and steric requirements. Back calculations of NOE intensities led us to introduce 535 additional distance constraints. Finally, 21 structures were selected as representative of the structure of the protein. The polypeptide backbone folds into a simple and original right-handed winding. It is composed of a bundle of four helices linked by flexible loops, which is packed against a C-terminal fragment forming a non-standard saxophone-like shape. The folded protein is stabilized by hydrophobic interactions and the four disulfide bridges combined by pairs on each side of the protein. An hydrophobic cleft, formed by residues located in the second half of the protein could be a potential site for the binding of lipids.


Assuntos
Proteínas de Transporte/química , Espectroscopia de Ressonância Magnética , Dobramento de Proteína , Triticum , Sequência de Aminoácidos , Antígenos de Plantas , Fenômenos Químicos , Físico-Química , Simulação por Computador , Dissulfetos/química , Eletroquímica , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas , Estrutura Secundária de Proteína , Termodinâmica
4.
Biochimie ; 76(2): 141-51, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8043650

RESUMO

The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.


Assuntos
Óperon Lac , Fatores de Lactose/química , Proteínas Repressoras/química , Sequência de Bases , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular
5.
Biochimie ; 74(9-10): 825-36, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1467342

RESUMO

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.


Assuntos
Defensinas , Hormônios de Inseto/química , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos , Peptídeos/química , Proteínas de Transferência de Fosfolipídeos , Proteínas/química , Sequência de Aminoácidos , Anti-Infecciosos/química , Proteínas de Bactérias/química , Proteínas de Transporte/química , Lipopeptídeos , Proteínas de Membrana/química , Dados de Sequência Molecular , Proteínas de Plantas/química , Conformação Proteica , Soluções , Relação Estrutura-Atividade , Tensoativos/química
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