RESUMO
Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.
Assuntos
Citocromos f/química , Plastocianina/química , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Método de Monte Carlo , Nostoc/química , Ressonância Magnética Nuclear Biomolecular , Eletricidade EstáticaRESUMO
Protein complex formation is thought to be at least a two-step process, in which the active complex is preceded by the formation of an encounter complex. The interactions in the encounter complex are usually dominated by electrostatic forces, whereas the active complex is also stabilized by noncovalent short-range forces. Here, the complex of cytochrome f and plastocyanin, electron-transfer proteins involved in photosynthesis, was studied using paramagnetic relaxation NMR spectroscopy. Spin labels were attached to cytochrome f, and the relaxation enhancements of plastocyanin nuclei were measured, demonstrating that a large part of the cytochrome f surface area is sampled by plastocyanin. In contrast, plastocyanin is always oriented with its hydrophobic patch toward cytochrome f. The complex was visualized using ensemble docking, showing that the encounter complex is stabilized by hydrophobic as well as electrostatic interactions. The results suggest a model of electrostatic preorientation before the proteins make contact, followed by the formation of an encounter complex that rapidly leads to electron-transfer active conformations by gradual increase of the overlap of nonpolar surface areas on cytochrome f and plastocyanin. In this model the distinction between the encounter and active complexes vanishes, at least in the case of electron-transfer complexes, which do not require a high degree of specificity.
Assuntos
Citocromos f/química , Ressonância Magnética Nuclear Biomolecular , Plastocianina/química , Citocromos f/isolamento & purificação , Citocromos f/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Método de Monte Carlo , Plastocianina/metabolismoRESUMO
Cytochrome f (Cyt f) and plastocyanin (Pc) form a highly transient complex as part of the photosynthetic redox chain. The complex from Nostoc sp. PCC 7119 was studied by NMR relaxation spectroscopy with the aim of determining the orientation of Pc relative to Cyt f. Chemical-shift-perturbation analysis showed that the presence of spin labels on the surface of Cyt f does not significantly affect the binding of Pc. The paramagnetic relaxation enhancement results are not consistent with a single orientation of Pc, thus indicating that multiple orientations must occur and suggesting that an encounter state represents a large fraction of the complex.
Assuntos
Citocromos f/metabolismo , Nostoc , Plastocianina/metabolismo , Citocromos f/química , Mesilatos/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Plastocianina/química , Ligação Proteica , Conformação ProteicaRESUMO
In photosynthetic organisms, ferredoxin (Fd) interacts with many proteins, acting as a shuttle for electrons from Photosystem I to a group of enzymes involved in NADP(+) reduction, sulfur and nitrogen assimilation, and the regulation of carbon assimilation. The study of the dynamic interactions between ferredoxin and these enzymes by nuclear magnetic resonance is severely hindered by the paramagnetic [2Fe-2S] cluster of a ferredoxin. To establish whether ferredoxin in which the cluster has been replaced by Ga is a suitable diamagnetic mimic, the solution structure of Synechocystis Ga-substituted ferredoxin has been determined and compared with the structure of the native protein. The ensemble of 10 structures with the lowest energies has an average root-mean-square deviation of 0.30 +/- 0.05 A for backbone atoms and 0.65 +/- 0.04 A for all heavy atoms. Comparison of the NMR structure of GaFd with the crystal structure of the native Fd indicates that the general structural fold found for the native, iron-containing ferredoxin is conserved in GaFd. The ferredoxin contains a single gallium and no inorganic sulfide. The distortion of the metal binding loop caused by the single gallium substitution is small. The binding site on Fd for binding ferredoxin:NADP(+) reductase in solution, determined using GaFd, includes the metal binding loop and its surroundings, consistent with the crystal structures of related complexes. The results provide a structural justification for the use of the gallium-substituted analogue in interaction studies.
Assuntos
Proteínas de Bactérias/química , Ferredoxinas/química , Gálio/química , Synechocystis/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ferredoxinas/metabolismo , Gálio/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Relação Estrutura-AtividadeRESUMO
Binding of fluorine-containing drugs to bovine beta-lactoglobulin, the most abundant whey protein in bovine milk, was investigated by means of (19)F NMR and mass spectrometry. The stoichiometry of the binding and its stability in acidic medium, where beta-lactoglobulin is folded and stable, were also studied, along with competition from molecules that can be regarded as analogs of physiological ligands to bovine beta-lactoglobulin. Conditional binding data were combined with protein structural information derived from circular dichroism and limited proteolysis studies. Spectroscopic techniques were also used to assess whether the bound drugs stabilize the protein structure against denaturation by chaotropes or temperature at various pH values. The results obtained provide evidence for the presence of multiple binding regions on the protein, with a specific and different affinity for structurally different classes of hydrophobic drugs and, more generally, that bovine beta-lactoglobulin can bind and protect against low pH values various classes of drugs of pharmaceutical relevance.
Assuntos
Proteínas de Transporte/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Indóis/metabolismo , Lactoglobulinas/metabolismo , Animais , Sítios de Ligação , Bovinos , Flúor , Flurbiprofeno/metabolismo , Fluvastatina , Concentração de Íons de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação ProteicaRESUMO
Many natural proteins are, as a whole or in part, intrinsically disordered. Frequently, such intrinsically disordered regions (IDRs) undergo a transition to a defined and often helical conformation upon binding to partner molecules. The intrinsic propensity of an IDR sequence to fold into a helical conformation already in the absence of a binding partner can have a decisive influence on the binding process and affinity. Using a combination of NMR spectroscopy and molecular dynamics (MD) simulations we have investigated the tendency of regions of Axin-1, an intrinsically disordered scaffolding protein of the WNT signaling pathway, to form helices in segments interacting with binding partners. Secondary chemical shifts from NMR measurements show an increased helical population in these regions. Systematic application of MD advanced sampling approaches on peptide segments of Axin-1 reproduces the experimentally observed tendency and allows insights into the distribution of segment conformations and free energies of helix formation. The results, however, were found to dependent on the force field water model. Recent water models specifically designed for IDRs significantly reduce the predicted helical content and do not improve the agreement with experiment.
Assuntos
Proteína Axina/química , Humanos , Ligação de Hidrogênio , Proteínas Intrinsicamente Desordenadas , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , TermodinâmicaRESUMO
The mammalian glutathione peroxidase (GPx) family is a key component of the cellular antioxidative defence system. Within this family, GPx4 has unique features as it accepts a large class of hydroperoxy lipid substrates and has a plethora of biological functions, including sperm maturation, regulation of apoptosis and cerebral embryogenesis. In this paper, the structure of the cytoplasmic isoform of mouse phospholipid hydroperoxide glutathione peroxidase (O70325-2 GPx4) with selenocysteine 46 mutated to cysteine is reported solved at 1.8â Å resolution using X-ray crystallography. Furthermore, solution data of an isotope-labelled GPx protein are presented.
Assuntos
Cisteína/química , Glutationa Peroxidase/química , Selenocisteína/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalografia por Raios X , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Camundongos , Modelos Moleculares , Mutação , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Plasmídeos/química , Plasmídeos/metabolismo , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Selenocisteína/metabolismo , Especificidade por SubstratoRESUMO
The combined use of in vitro (19F-NMR) and in silico (molecular docking) procedures demonstrates the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine) for different drugs (mainly steroids and vastatins). Comparative evaluations on the complexes outline some of the features relevant for interaction (non-polar character of the drugs; amino acids and water molecules in the protein calyx most often involved in binding). Dissociation constants (Ki) for drugs typically lie in the same range as Ki for natural ligands; in most instances (different proteins and docking conditions), vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. The possible biological implications of this feature are discussed in connection with drug delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues).
Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Animais , Ligação Competitiva , Galinhas , Simulação por Computador , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos , Interações Medicamentosas , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/isolamento & purificação , Humanos , Técnicas In Vitro , Ligantes , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Conformação ProteicaRESUMO
Electron transfer proteins transport electrons safely between large redox enzymes. The complexes formed by these proteins are among the most transient. The biological function requires, on the one hand, sufficient specificity of the interaction to allow for rapid and selective electron transfer, and, on the other hand, a fast turnover of the complex. Recent progress in the characterization of the nature of these complexes has demonstrated that the encounter state plays an important role. This state of initial binding is dominated by electrostatic interactions, and consists of an ensemble of orientations. Paramagnetic relaxation enhancement NMR and chemical shift perturbation analysis provide ways for the experimental characterisation of the encounter state. Several studies that have used these techniques have shown that the surface area sample in the encounter state can be limited to the immediate environment of the final, specific complex. The encounter complex can represent a large fraction and, in some small complexes, no specific binding is detected at all. It can be concluded that, in electron transfer protein complexes, a fine balance is sought between the low-specificity encounter state and the high-specificity productive complex to meet the opposing requirements of rapid electron transfer and a high turnover rate.