RESUMEN
A nonsense suppression method was employed to incorporate a total of four natural and six unnatural residues at the 9' position of the M2 region in the beta, gamma, and delta subunits of muscle nicotinic receptors. In 33 pairwise comparisons of functional properties as influenced by structural features including side chain length, branching, and substitution of oxygen for methylene carbons, it is concluded that increased polarity in the side chains at the 9' position consistently increases the sensitivity to acetylcholine. In addition, the stereochemistry of the side chain can have marked influences on the EC50, primarily because of changes in the single-channel open time. For the case of isoleucine versus allo-isoleucine in the delta subunit, these changes are themselves modified by mutations at the 9' position in other subunits. The data suggest an especially strong interaction between the beta and delta subunits in the pore region, leading in turn to a suggested arrangement of subunits within the pentamer.
Asunto(s)
Activación del Canal Iónico , Canales Iónicos/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Animales , Femenino , Oocitos , Estereoisomerismo , Factores de Tiempo , XenopusRESUMEN
Structure-function relations in the nicotinic acetylcholine receptor are probed using a recently developed method based on chemical synthesis of nonsense suppressor tRNAs with unnatural amino acid residues, site-directed incorporation at nonsense codons in Xenopus laevis oocytes, and electrophysiological measurements. A broad range of unnatural amino acids, as many as 14 at a given site, are incorporated at three sites, alpha 93, alpha 190, and alpha 198, all of which are tyrosine in the wild-type receptor and are thought to contribute to the agonist binding site. Confirming and expanding upon earlier studies using conventional mutagenesis, the three tyrosines are shown to be in substantially different structural microenvironments. In particular, a crucial role is established for the hydroxyl group of alpha Tyr93, whereas a variety of substituents are functional at the analogous position of alpha Tyr198. Interestingly, consideration of three different agonists (acetylcholine, nicotine, and tetramethylammonium) does not discriminate between these two best-characterized binding site residues. In addition, double-mutation studies establish the independent effects of mutations at the pore region (second transmembrane region) and at the agonist binding site, and this observation leads to a novel strategy for adjusting EC50 values. These results establish the broad generality and great potential of the unnatural amino acid methodology for illuminating subtle structural distinctions in neuroreceptors and related integral membrane proteins.
Asunto(s)
Aminoácidos/química , Aminoácidos/metabolismo , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Aminoácidos/farmacología , Animales , Sitios de Unión , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Agonistas Nicotínicos/metabolismo , Compuestos de Amonio Cuaternario/farmacología , Receptores Nicotínicos/genética , Relación Estructura-Actividad , Xenopus laevisRESUMEN
A new tRNA, THG73, has been designed and evaluated as a vehicle for incorporating unnatural amino acids site-specifically into proteins expressed in vivo using the stop codon suppression technique. The construct is a modification of tRNAGln(CUA) from Tetrahymena thermophila, which naturally recognizes the stop codon UAG. Using electrophysiological studies of mutations at several sites of the nicotinic acetylcholine receptor, it is established that THG73 represents a major improvement over previous nonsense suppressors both in terms of efficiency and fidelity of unnatural amino acid incorporation. Compared with a previous tRNA used for in vivo suppression, THG73 is as much as 100-fold less likely to be acylated by endogenous synthetases of the Xenopus oocyte. This effectively eliminates a major concern of the in vivo suppression methodology, the undesirable incorporation of natural amino acids at the suppression site. In addition, THG73 is 4-10-fold more efficient at incorporating unnatural amino acids in the oocyte system. Taken together, these two advances should greatly expand the range of applicability of the in vivo nonsense suppression methodology.
Asunto(s)
Aminoácidos/metabolismo , Codón sin Sentido , ARN de Transferencia de Glutamina/genética , Receptores Colinérgicos/genética , Supresión Genética , Tetrahymena thermophila/genética , Acetilcolina/farmacología , Acilación , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Conductividad Eléctrica , Ingeniería Genética , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Receptores Colinérgicos/biosíntesis , Estereoisomerismo , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.
Asunto(s)
Fenilalanina/química , Receptores Nicotínicos/metabolismo , Tirosina/química , Animales , Secuencia de Bases , Sitios de Unión , Codón , Enlace de Hidrógeno , Ligandos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos , Fenilalanina/análogos & derivados , Receptores Nicotínicos/química , Relación Estructura-Actividad , Tirosina/análogos & derivados , XenopusRESUMEN
Restriction maps of two plasmids encoding parathion hydrolase have been determined. pPDL2 is a 39-kb plasmid harbored by Flavobacterium sp. (ATCC 27551), while pCMS1 is a 70-kb plasmid found in Pseudomonas diminuta (strain MG). Both plasmids previously have been shown to share homologous parathion hydrolase genes (termed opd for organophosphate degradation) as judged by DNA-DNA hybridization and restriction mapping. In the present study, we conducted DNA hybridization experiments using each of nine PstI restriction fragments from pCMS1 as probes against Flavobacterium plasmid DNA. The opd genes of both plasmids are located within a highly conserved region of approximately 5.1 kb. This region of homology extends approximately 2.6 kb upstream and 1.7 kb downstream from the opd genes. No homology between the two plasmids is evident outside of this region.
Asunto(s)
Flavobacterium/genética , Monoéster Fosfórico Hidrolasas/genética , Plásmidos , Pseudomonas/genética , Arildialquilfosfatasa , Enzimas de Restricción del ADN , Flavobacterium/enzimología , Hibridación de Ácido Nucleico , Mapeo Nucleótido , Pseudomonas/enzimologíaRESUMEN
Parathion hydrolases have been previously described for an American isolate of Pseudomonas diminuta and a Philippine isolate of Flavobacterium sp. (ATCC 27551). The gene which encodes the broad-spectrum organophosphate phosphotriesterase in P. diminuta has been shown by other investigators to be located on a 66-kilobase (kb) plasmid. The intact gene (opd, organophosphate-degrading gene) from this degradative plasmid was cloned into M13mp10 and found to express parathion hydrolase under control of the lac promoter in Escherichia coli. In Flavobacterium sp. strain ATCC 27551, a 43-kb plasmid was associated with the production of parathion hydrolase by curing experiments. The M13mp10-cloned fragment of the opd gene from P. diminuta was used to identify a homologous genetic region from Flavobacterium sp. strain ATCC 27551. Southern hybridization experiments demonstrated that a genetic region from the 43-kb Flavobacterium sp. plasmid possessed significant homology to the opd sequence. Similar hybridization did not occur with three other native Flavobacterium sp. plasmids (approximately 23, 27, and 51 kb) present within this strain or with genomic DNA from cured strains. Restriction mapping of various recombinant DNA molecules containing subcloned fragments of both opd plasmids revealed that the restriction maps of the two opd regions were similar, if not identical, for all restriction endonucleases tested thus far. In contrast, the restriction maps of the cloned plasmid sequences outside the opd regions were not similar. Thus, it appears that the two discrete bacterial plasmids from parathion-hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures.
Asunto(s)
Flavobacterium/enzimología , Paratión/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Plásmidos , Pseudomonas/genética , Arildialquilfosfatasa , Biodegradación Ambiental , Clonación Molecular , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Escherichia coli/genética , Flavobacterium/genética , Genes Bacterianos , Operón Lac , Hibridación de Ácido Nucleico , Monoéster Fosfórico Hidrolasas/metabolismo , Pseudomonas/enzimología , Homología de Secuencia de Ácido NucleicoAsunto(s)
Atrazina/análogos & derivados , Nitrosaminas , Atrazina/metabolismo , Fenómenos Químicos , Química , Ecología , Nitrosaminas/metabolismo , SueloRESUMEN
Experiments to grow Tetraselmis chuii (a marine alga) and Daphnia magna in the presence of inorganic arsenate are described. The algae incorporate arsenic rather efficiently and form a lipid-soluble organic arsenic compound. T. chuii has been successfully mass cultured in a medium containing 10 ppm arsenic as arsenate. Daphnia magna was cultured in a medium containing 74As-labeled H3AsO4 and 1 ppm Na2HAsO4 expressed as arsenic. The arsenic metabolites were extracted with a chloroform-methanol solution and isolated by using column and thin-layer chromatography. TLC analysis of the metabolites revealed the presence of a 74As-containing product which migrated with phosphatidylethanolamine. This product was hydrolyzed with the phospholipases A, C, and D. The experimental results are not inconsistent with the presence of an arsenocholine moiety in the lipids. Arsenocholine, arsenobetaine, and acetylarsenocholine have been synthesized and will serve as reference substances in the chromatography experiments. The preparation of arsenocholine-containing lipids is in progress.