Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 20
Filtrar
1.
Nature ; 538(7625): 329-335, 2016 Oct 20.
Artículo en Inglés | MEDLINE | ID: mdl-27626386

RESUMEN

Naturally occurring, pharmacologically active peptides constrained with covalent crosslinks generally have shapes that have evolved to fit precisely into binding pockets on their targets. Such peptides can have excellent pharmaceutical properties, combining the stability and tissue penetration of small-molecule drugs with the specificity of much larger protein therapeutics. The ability to design constrained peptides with precisely specified tertiary structures would enable the design of shape-complementary inhibitors of arbitrary targets. Here we describe the development of computational methods for accurate de novo design of conformationally restricted peptides, and the use of these methods to design 18-47 residue, disulfide-crosslinked peptides, a subset of which are heterochiral and/or N-C backbone-cyclized. Both genetically encodable and non-canonical peptides are exceptionally stable to thermal and chemical denaturation, and 12 experimentally determined X-ray and NMR structures are nearly identical to the computational design models. The computational design methods and stable scaffolds presented here provide the basis for development of a new generation of peptide-based drugs.


Asunto(s)
Diseño Asistido por Computadora , Diseño de Fármacos , Péptidos/química , Péptidos/síntesis química , Estabilidad Proteica , Secuencias de Aminoácidos , Cristalografía por Rayos X , Ciclización , Disulfuros/química , Calor , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Péptidos/genética , Péptidos Cíclicos/química , Péptidos Cíclicos/genética , Desnaturalización Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Estereoisomerismo
2.
Biochemistry ; 57(5): 511-515, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29251491

RESUMEN

Cullin-ring-ligases mediate protein polyubiquitination, a signal for degradation in the 26S proteasome. The CRL1 class consists of Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF complex, the 162-amino acid DdSkp1 from Dictyostelium bridges cullin-1 with an F-box protein (FBP), the specificity factor for substrate selection. The hydroxylation-dependent glycosylation of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel O2-sensing mechanism in the social amoeba Dictyostelium and other protists. Previous evidence indicated that glycosylation promotes increased α-helical content correlating with enhanced interaction with three F-box proteins. To localize these differences, we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We report NMR assignments of backbone 1HN, 15N, 13Cα, and 13CO nuclei as well as side-chain 13Cß and methyl 13C/1H nuclei of Ile(δ1), Leu, and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index and 15N{1H} HNOE indicate that the C-terminal region, which forms a helix-loop-helix motif centered on Pro143 at the crystallographically defined binding interface with F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical shifts indicate that the variation of conformation in Gn-DdSkp1, relative to DdSkp1, is limited to this region and characterized by increased helical fold. Extension of the glycan chain results in further changes, also limited to this region. Thus, glycosylation may control F-box protein interactions via a local effect on DdSkp1 conformation, by a mechanism that may be general to many unicellular eukaryotes.


Asunto(s)
Dictyostelium/metabolismo , Secuencias F-Box , Proteínas F-Box/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Secuencia de Aminoácidos , Dictyostelium/química , Proteínas F-Box/química , Glicosilación , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Mapas de Interacción de Proteínas , Proteínas Protozoarias/química , Proteínas Quinasas Asociadas a Fase-S/química , Proteínas Ligasas SKP Cullina F-box/química , Proteínas Ligasas SKP Cullina F-box/metabolismo , Alineación de Secuencia
3.
Int J Syst Evol Microbiol ; 65(11): 4146-4150, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26303654

RESUMEN

A Gram-stain-positive, aerobic, motile and non-spore-forming actinobacterium, strain Y32T, was isolated from a deep-sea sediment of the western Pacific Ocean. Phylogenetic and phenotypic properties of the organism supported that it belonged to the genus Georgenia. Strain Y32T shared highest 16S rRNA gene sequence similarity of 97.8 % with Georgenia muralis 1A-CT, followed by Georgenia thermotolerans TT02-04T (97.4 %), Georgenia daeguensis 2C6-43T (97.2 %), Oceanitalea nanhaiensis JLT1488T (97.2 %), Georgenia ruanii YIM 004T (97.0 %) and Georgenia soli CC-NMPT-T3T (97.0 %). The organism grew in the presence of 0-10 % (w/v) NaCl, at 4-40 °C and at pH 6-11, with optimal growth occurring at 30-35 °C, at pH 7 and in the presence of 3.5 % (w/v) NaCl. The polar lipid profile of strain Y32T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and two phosphatidylinositol mannosides. Strain Y32T contained MK-8(H4) and MK-7(H4) as the major components of the menaquinone system, and anteiso-C15 : 0, iso-C15 : 0 and iso-C14 : 0 as the predominant fatty acids. Galactose was detected as the cell-wall sugar. The G+C content of the DNA was 71.2 mol%. Based on the results of phenotypic, genotypic and phylogenetic analyses, it is considered that strain Y32T represents a novel species of the genus Georgenia, for which the name Georgenia subflava sp. nov. is proposed. The type strain is Y32T ( = LMG 28101T = CGMCC 1.12782T = JCM 19765T = MCCC 1A09955T).


Asunto(s)
Actinomycetales/clasificación , Sedimentos Geológicos/microbiología , Filogenia , Agua de Mar/microbiología , Actinomycetales/genética , Actinomycetales/aislamiento & purificación , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Océano Pacífico , Fosfolípidos/química , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Vitamina K 2/análogos & derivados , Vitamina K 2/química
4.
J Struct Funct Genomics ; 15(4): 201-7, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24941917

RESUMEN

High-quality solution NMR structures of three homeodomains from human proteins ALX4, ZHX1 and CASP8AP2 were solved. These domains were chosen as targets of a biomedical theme project pursued by the Northeast Structural Genomics Consortium. This project focuses on increasing the structural coverage of human proteins associated with cancer.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/química , Proteínas de Unión al Calcio/química , Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , Proteínas de Neoplasias/química , Neoplasias/química , Factores de Transcripción/química , Humanos , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína
5.
Biochemistry ; 52(34): 5800-8, 2013 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-23906368

RESUMEN

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²âº-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²âº-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²âº-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²âº-free recoverin and 3.7 ± 0.1 nm for Ca²âº-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²âº-bound recoverin may represent an intermolecular distance between C39 and N120. ¹5N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²âº-bound recoverin forms a dimer at protein concentrations above 100 µM, whereas Ca²âº-free recoverin is monomeric. We propose that Ca²âº-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²âº-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²âº-induced dimerization is functionally important but not well understood.


Asunto(s)
Calcio/farmacología , Conformación Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Recoverina/química , Espectroscopía de Resonancia por Spin del Electrón , Electrones , Espectroscopía de Resonancia Magnética , Ácidos Mirísticos/metabolismo , Recoverina/metabolismo , Marcadores de Spin
6.
Proc Natl Acad Sci U S A ; 107(44): 19102-7, 2010 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-20956299

RESUMEN

Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the female's antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBP-mediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP·pheromone complex. Although CquiOBP1 binds MOP in a pH-dependent fashion, it lacks the C terminus required for the pH-dependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.


Asunto(s)
Culex/química , Proteínas de Insectos/química , Modelos Moleculares , Pironas/química , Receptores Odorantes/química , Atractivos Sexuales/química , Animales , Cristalografía por Rayos X , Culex/metabolismo , Femenino , Concentración de Iones de Hidrógeno , Proteínas de Insectos/metabolismo , Oviposición/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Pironas/metabolismo , Receptores Odorantes/metabolismo , Atractivos Sexuales/metabolismo , Relación Estructura-Actividad
7.
Proteins ; 79(6): 1910-22, 2011 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-21465563

RESUMEN

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, serves as a calcium sensor in retinal rod cells. Ca(2+) -induced conformational changes in recoverin promote extrusion of its covalently attached myristate, known as the Ca(2+)-myristoyl switch. Here, we present nuclear magnetic resonance (NMR) relaxation dispersion and chemical shift analysis on (15) N-labeled recoverin to probe main chain conformational dynamics. (15) N NMR relaxation data suggest that Ca(2+)-free recoverin undergoes millisecond conformational dynamics at particular amide sites throughout the protein. The addition of trace Ca(2+) levels (0.05 equivalents) increases the number of residues that show detectable relaxation dispersion. The Ca(2+)-dependent chemical shifts and relaxation dispersion suggest that recoverin has an intermediate conformational state (I) between the sequestered apo state (T) and Ca(2+) saturated extruded state (R): T ↔ I ↔ R. The first step is a fast conformational equilibrium ([T]/[I] < 100) on the millisecond time scale (τ(ex) δω < 1). The final step (I ↔ R) is much slower (τ(ex) δω > 1). The main chain structure of I is similar in part to the structure of half-saturated E85Q recoverin with a sequestered myristoyl group. We propose that millisecond dynamics during T ↔ I may transiently increase the exposure of Ca(2+)-binding sites to initiate Ca(2+) binding that drives extrusion of the myristoyl group during I ↔ R.


Asunto(s)
Calcio/metabolismo , Ácido Mirístico/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Recoverina/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Modelos Moleculares , Datos de Secuencia Molecular , Isótopos de Nitrógeno/química , Conformación Proteica , Recoverina/química , Alineación de Secuencia
8.
Biochem Biophys Res Commun ; 404(1): 335-8, 2011 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-21130734

RESUMEN

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present a mutational analysis on a PBP from A. transitella (AtraPBP1) to evaluate how the C-terminal helix in this protein controls pheromone binding as a function of pH. Pheromone binds tightly to AtraPBP1 at neutral pH, but the binding is much weaker at pH below 5. Deletion of the entire C-terminal helix (residues 129-142) causes more than 100-fold increase in pheromone-binding affinity at pH 5 and only a 1.5-fold increase at pH 7. A similar pH-dependent increase in pheromone binding is also seen for the H80A/H95A double mutant that promotes extrusion of the C-terminal helix by disabling salt bridges at each end of the helix. The single mutants (H80A and H95A) also exhibit pheromone binding at pH below 5, but with ∼2-fold weaker affinity. NMR and circular dichroism data demonstrate a large overall structural change in each of these mutants at pH 4.5, indicating an extrusion of the C-terminal helix that profoundly affects the overall structure of the low pH form. Our results confirm that sequestration of the C-terminal helix at low pH as seen in the recent NMR structure may serve to block pheromone binding. We propose that extrusion of these C-terminal residues at neutral pH (or by the mutations in this study) exposes a hydrophobic cleft that promotes high affinity pheromone binding.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Insectos/química , Mariposas Nocturnas/metabolismo , Feromonas/química , Animales , Proteínas Portadoras/genética , Dicroismo Circular , Análisis Mutacional de ADN , Femenino , Concentración de Iones de Hidrógeno , Proteínas de Insectos/genética , Masculino , Mutación , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
9.
Biochemistry ; 49(7): 1469-76, 2010 Feb 23.
Artículo en Inglés | MEDLINE | ID: mdl-20088570

RESUMEN

The navel orangeworm, Amyelois transitella (Walker), is an agricultural insect pest that can be controlled by disrupting male-female communication with sex pheromones, a technique known as mating disruption. Insect pheromone-binding proteins (PBPs) provide fast transport of hydrophobic pheromones through the aqueous sensillar lymph and promote sensitive delivery of pheromones to receptors. Here we present the three-dimensional structure of a PBP from A. transitella (AtraPBP1) in solution at pH 4.5 determined by nuclear magnetic resonance (NMR) spectroscopy. Pulsed-field gradient NMR diffusion experiments, multiangle light scattering, and (15)N NMR relaxation analysis indicate that AtraPBP1 forms a stable monomer in solution at pH 4.5 in contrast to forming mostly dimers at pH 7. The NMR structure of AtraPBP1 at pH 4.5 contains seven alpha-helices (alpha1, L8-L23; alpha2, D27-F36; alpha3, R46-V62; alpha4, A73-M78; alpha5, D84-S100; alpha6, R107-L125; alpha7, M131-E141) that adopt an overall main-chain fold similar to that of PBPs found in Antheraea polyphemus and Bombyx mori. The AtraPBP1 structure is stabilized by three disulfide bonds formed by C19/C54, C50/C108, and C97/C117 and salt bridges formed by H69/E60, H70/E57, H80/E132, H95/E141, and H123/D40. All five His residues are cationic at pH 4.5, whereas H80 and H95 become neutral at pH 7.0. The C-terminal helix (alpha7) contains hydrophobic residues (M131, V133, V134, V135, V138, L139, and A140) that contact conserved residues (W37, L59, A73, F76, A77, I94, V111, and V115) suggested to interact with bound pheromone. Our NMR studies reveal that acid-induced formation of the C-terminal helix at pH 4.5 is triggered by a histidine protonation switch that promotes rapid release of bound pheromone under acidic conditions.


Asunto(s)
Proteínas Portadoras/química , Proteínas de Insectos/química , Mariposas Nocturnas/química , Feromonas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Cristalografía por Rayos X , Femenino , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Insectos/metabolismo , Proteínas de Insectos/fisiología , Masculino , Datos de Secuencia Molecular , Mariposas Nocturnas/fisiología , Resonancia Magnética Nuclear Biomolecular , Feromonas/metabolismo , Feromonas/fisiología , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Conducta Sexual Animal
10.
Ecotoxicol Environ Saf ; 73(6): 1196-201, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20576290

RESUMEN

To gain insight into the reproductive toxicity of sublethal cadmium on marine polychaetes, Perinereis nuntia sandworms were exposed to cadmium via artificially contaminated seawater. Cadmium influence on sexual maturation, egg laying, fertilization, zygote hatching and vitellogenin expression levels were analyzed. Results indicated that 23.05 and 563.87 microg L(-1) cadmium significantly delayed sexual maturation. Fertilization rate was significantly inhibited by 563.87 microg L(-1) cadmium while lower concentrations showed no significant effect. Zygote hatching was significantly inhibited by cadmium concentrations greater than 1.12 microg L(-1). We identified a vitellogenin gene sequence in P. nuntia and found that mRNA transcription was significantly upregulated by cadmium. These results indicate that sublethal cadmium levels cause dose-dependent reproductive toxicity on P. nuntia by inhibiting sexual maturation, fertilization and zygote hatching, and the increased expression of vitellogenin suggesting cadmium has strong feminization effects on polychaetes.


Asunto(s)
Cadmio/toxicidad , Poliquetos/efectos de los fármacos , Agua de Mar/química , Contaminantes Químicos del Agua/toxicidad , Análisis de Varianza , Animales , Femenino , Expresión Génica/efectos de los fármacos , Larva/efectos de los fármacos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Masculino , Poliquetos/genética , Poliquetos/crecimiento & desarrollo , Poliquetos/metabolismo , Reproducción/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Maduración Sexual/efectos de los fármacos , Pruebas de Toxicidad , Vitelogeninas/genética
11.
Mol Biochem Parasitol ; 201(1): 72-75, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-26099941

RESUMEN

Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca(2+) sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca(2+) binding. It is similar to neuronal Ca(2+) sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca(2+)-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca(2+), permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca(2+), disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca(2+)-binding mutant FCaBP(E151Q/E188Q) and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP(E151Q/E188Q) were found to associate with lipid rafts, indicating the Ca(2+)-independence of this association. To our initial surprise, FCaBP(E151Q/E188Q), like wildtype FCaBP, exhibited Ca(2+)-dependent flagellar membrane association, even though this protein does not bind Ca(2+) itself [16]. One possible explanation for this is that FCaBP(E151Q/E188Q), like some other Ca(2+) sensors, may form dimers and that dimerization of FCaBP(E151Q/E188Q) with endogenous wildtype FCaBP might explain its Ca(2+)-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca(2+). These results suggest that FCaBP possesses two distinct Ca(2+)-dependent interactions-one involving a Ca(2+)-induced change in conformation and another perhaps involving binding partner association.


Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Flagelos/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Microdominios de Membrana/química , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética
12.
Biomol NMR Assign ; 7(1): 9-12, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22382573

RESUMEN

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of Trypanosoma brucei. (BMRB no. 18011).


Asunto(s)
Proteínas de Unión al Calcio/química , Flagelos , Resonancia Magnética Nuclear Biomolecular , Proteínas Protozoarias/química , Trypanosoma brucei brucei/citología , Secuencia de Aminoácidos , Datos de Secuencia Molecular
13.
Protein Sci ; 21(12): 1942-7, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23011904

RESUMEN

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF-hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF-hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N-terminal myristoyl group may be important for membrane binding. Non-conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.


Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Secuencia de Aminoácidos , Enfermedad de Chagas/parasitología , Motivos EF Hand , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Alineación de Secuencia , Trypanosoma cruzi/química , Tripanosomiasis Africana/parasitología
14.
Structure ; 18(1): 9-16, 2010 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-20152148

RESUMEN

Perhaps 5%-10% of proteins bind to membranes via a covalently attached lipid. Posttranslational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca(2+) signaling pathway in vision, and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol-4,5-bisphosphate. Both proteins have been successfully encapsulated in the lipid-extruded state and high-resolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.


Asunto(s)
Proteínas de la Membrana/análisis , Micelas , Resonancia Magnética Nuclear Biomolecular/métodos , Cápsulas/química , Ácidos Grasos/química , Antígenos VIH/análisis , Antígenos VIH/química , VIH-1/química , Proteínas de la Membrana/química , Recoverina/análisis , Recoverina/química , Solubilidad , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/análisis , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/química
15.
PLoS One ; 4(9): e7235, 2009 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-19789654

RESUMEN

BACKGROUND: The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control--like pheromone-based approaches--are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. METHODOLOGY: By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. CONCLUSION: We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH.


Asunto(s)
Mariposas Nocturnas/fisiología , Receptores Odorantes/metabolismo , Olfato/genética , Animales , Secuencia de Bases , Dicroismo Circular , Clonación Molecular , Insectos , Espectroscopía de Resonancia Magnética/métodos , Microscopía Fluorescente/métodos , Datos de Secuencia Molecular , Mariposas Nocturnas/metabolismo , Filogenia , Conformación Proteica , Estructura Terciaria de Proteína , Atractivos Sexuales/metabolismo
16.
Biomol NMR Assign ; 3(2): 195-7, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19888689

RESUMEN

An odorant-binding protein from the Southern house mosquito, Culex pipiens quinquefasciatus (Cqui-OBP1) binds to the mosquito oviposition pheromone (MOP), 6-acetoxy-5-hexadecanolide to facilitate the transport of MOP to membrane-bound odorant receptors. We report complete NMR chemical shift assignments of Cqui-OBP1 bound to the MOP pheromone obtained at pH 7.0 and 25 degrees C (BMRB no. 16175).


Asunto(s)
Culicidae , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Oviposición , Feromonas/metabolismo , Receptores Odorantes/química , Receptores Odorantes/metabolismo , Animales , Culicidae/fisiología , Concentración de Iones de Hidrógeno , Resonancia Magnética Nuclear Biomolecular
17.
Ying Yong Sheng Tai Xue Bao ; 19(9): 2087-91, 2008 Sep.
Artículo en Zh | MEDLINE | ID: mdl-19102328

RESUMEN

The study showed that 2 450 MHz microwave irradiation for 35 seconds or more had significant death effects on Moina mongolica Daday. Short-term (less than 25 seconds) microwave irradiation could obviously increase the larvae number per clutch and the total fecundity over life span of the animal, with the highest fecundity under 10 seconds irradiation. Microwave irradiation could significantly prolong the life span (15.8-18 d) of M. mongolica. The short-term microwave irradiation had less effect on the development of larvae animal, but inhibited the adult growth to some degree. Impulse electromagnetic field could significantly increase the fecundity of M. mongolica, with the highest effect of 29 kV x cm(-1); while high-voltage electrostatic field had less effect on the reproduction of M. mongolica. Both high-voltage and impulse electrostatic fields had no remarkable effects on the development of larvae animal. High-voltage electrostatic field had less effect on the growth of adult animal; while impulse electromagnetic field had definite inhibition effect on it, and the inhibition effect was increased with increasing voltage.


Asunto(s)
Cladóceros/efectos de la radiación , Campos Electromagnéticos , Microondas , Animales , Cladóceros/crecimiento & desarrollo , Cladóceros/fisiología , Reproducción/efectos de la radiación
18.
Biomol NMR Assign ; 2(2): 105-6, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19636880

RESUMEN

A pheromone-binding protein from navel orange worm, Amyelois transitella (Atra-PBP1) binds to non-polar pheromone molecules and facilitates the transport and delivery of pheromone to the membrane-bound pheromone receptors. We report complete NMR chemical shift assignments of Atra-PBP1 obtained at pH 4.5 and 25 degrees C (BMRB No. 15601).


Asunto(s)
Proteínas Portadoras/química , Helmintos/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Secuencia de Aminoácidos , Animales , Isótopos de Carbono/química , Datos de Secuencia Molecular , Peso Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , Protones
19.
Biochemistry ; 45(10): 3404-11, 2006 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-16519535

RESUMEN

An earlier study shows that a 30 min incubation of spinach PS II submembrane fragments at pH 6.3 in the presence of 10 microM HgCl(2) induces a 40% depletion of the 33 kDa protein without the apparent release of the 17 and 23 kDa proteins [Bernier, M., and Carpentier, R. (1995) FEBS Lett. 360, 251-254]. Here we report that the photosystem II 33 kDa extrinsic protein is fully removed by HgCl(2) added at micromolar and higher concentrations (0.25, 20, and 50 microM), with the 17 and 23 kDa extrinsic proteins and other intrinsic proteins remaining bound to the reaction center. The data presented here put in doubt the "regulatory cap" model of PS II, which follows the OEC-33 kDa-23 kDa-17 kDa binding order, as these results directly demonstrate that the 33 kDa protein can be removed without affecting the binding of the 23 and 17 kDa proteins to the intrinsic subunits of PS II. This suggests that each extrinsic protein may possess its own binding site on PS II. A possible mechanism for HgCl(2) upon the release of the 33 kDa protein is discussed.


Asunto(s)
Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/metabolismo , Spinacia oleracea/metabolismo , Sitios de Unión , Cloruros/metabolismo , Cloruros/farmacología , Relación Dosis-Respuesta a Droga , Concentración de Iones de Hidrógeno , Cloruro de Mercurio/metabolismo , Cloruro de Mercurio/farmacología , Modelos Biológicos , Péptidos/química , Complejo de Proteína del Fotosistema II/química , Unión Proteica , Tilacoides/efectos de los fármacos , Tilacoides/metabolismo , Factores de Tiempo
20.
Biochemistry ; 44(36): 12022-9, 2005 Sep 13.
Artículo en Inglés | MEDLINE | ID: mdl-16142899

RESUMEN

The effect of adding azide to photosystem II (PS II) membrane samples (BBY preparation), with or without chloride, has been investigated using continuous wave (CW) and pulsed EPR spectroscopy. In the BBY samples with 25 mM chloride, we observed that the inhibition induced by azide is partly recovered by the addition of bicarbonate. Electron spin-echo envelope modulation (ESEEM) was used to search for spin transitions of 15N nuclei magnetically coupled to the S2 state Mn cluster (multiline EPR signal form) in 15N (single terminal label) azide-treated samples with negative results. However, an 15N ESEEM peak was observed in parallel chloride-depleted PS II samples when the 15N-labeled azide is added. However, this peak is absent in chloride-depleted samples incubated in buffer containing both chloride and [15N]azide. Thus these results demonstrate an azide binding site in the immediate vicinity of the Mn cluster, and since this site appears to be competitive with chloride, these results provide further evidence that chloride is bound proximal to the Mn cluster as well. Discussion on the possible interplay between azide, chloride, and bicarbonate is provided.


Asunto(s)
Azidas/metabolismo , Cloruros/metabolismo , Manganeso/metabolismo , Complejo de Proteína del Fotosistema II/química , Complejo de Proteína del Fotosistema II/metabolismo , Azidas/química , Sitios de Unión , Espectroscopía de Resonancia por Spin del Electrón , Iones/química , Spinacia oleracea/química , Spinacia oleracea/citología , Spinacia oleracea/enzimología , Tilacoides/química , Tilacoides/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA