RESUMEN
Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.
Asunto(s)
Bacteriorodopsinas/metabolismo , Tirosina/fisiología , Bacteriorodopsinas/genética , Halobacterium salinarum/química , Halobacterium salinarum/genética , Concentración de Iones de Hidrógeno , Cinética , Luz , Mutagénesis Sitio-Dirigida , Mutación , Fotólisis , Plásmidos , Protones , Tirosina/químicaRESUMEN
Cardiac hypertrophy is characterized by both remodeling of the extracellular matrix (ECM) and hypertrophic growth of the cardiocytes. Here we show increased expression and cytoskeletal association of the ECM proteins fibronectin and vitronectin in pressure-overloaded feline myocardium. These changes are accompanied by cytoskeletal binding and phosphorylation of focal adhesion kinase (FAK) at Tyr-397 and Tyr-925, c-Src at Tyr-416, recruitment of the adapter proteins p130(Cas), Shc, and Nck, and activation of the extracellular-regulated kinases ERK1/2. A synthetic peptide containing the Arg-Gly-Asp (RGD) motif of fibronectin and vitronectin was used to stimulate adult feline cardiomyocytes cultured on laminin or within a type-I collagen matrix. Whereas cardiocytes under both conditions showed RGD-stimulated ERK1/2 activation, only collagen-embedded cells exhibited cytoskeletal assembly of FAK, c-Src, Nck, and Shc. In RGD-stimulated collagen-embedded cells, FAK was phosphorylated only at Tyr-397 and c-Src association occurred without Tyr-416 phosphorylation and p130(Cas) association. Therefore, c-Src activation is not required for its cytoskeletal binding but may be important for additional phosphorylation of FAK. Overall, our study suggests that multiple signaling pathways originate in pressure-overloaded heart following integrin engagement with ECM proteins, including focal complex formation and ERK1/2 activation, and many of these pathways can be activated in cardiomyocytes via RGD-stimulated integrin activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Cardiomegalia/metabolismo , Integrinas/metabolismo , Integrinas/fisiología , Actinas/metabolismo , Animales , Western Blotting , Gatos , Células Cultivadas , Colágeno/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Activación Enzimática , Matriz Extracelular/patología , Fibronectinas/metabolismo , Proteína-Tirosina Quinasas de Adhesión Focal , Laminina/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocardio/metabolismo , Proteínas Oncogénicas/metabolismo , Péptidos/farmacología , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Transducción de Señal , Factores de Tiempo , Tirosina/química , Vitronectina/metabolismoRESUMEN
Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.
Asunto(s)
Arginina/genética , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Histidina/genética , Mutagénesis Sitio-Dirigida , Protones , Arginina/metabolismo , Ácido Aspártico/genética , Bacteriorodopsinas/metabolismo , Oscuridad , Halobacterium salinarum/química , Halobacterium salinarum/genética , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Luz , Fotólisis , Membrana Púrpura/química , Membrana Púrpura/metabolismoRESUMEN
Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 micros to 75 micros with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.
Asunto(s)
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Luz , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Protones , Bases de Schiff/metabolismo , Absorción , Adaptación Fisiológica , Sustitución de Aminoácidos , Bacteriorodopsinas/genética , Transporte Biológico , Color , Oscuridad , Espacio Extracelular/metabolismo , Halobacterium salinarum/citología , Halobacterium salinarum/metabolismo , Halobacterium salinarum/fisiología , Halobacterium salinarum/efectos de la radiación , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Proteínas Mutantes/genética , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Conformación Proteica , EstereoisomerismoRESUMEN
Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Microtúbulos/química , Paclitaxel/farmacología , Tubulina (Proteína)/fisiología , Animales , Células CHO , Cricetinae , Resistencia a Antineoplásicos , Leucina , Relación Estructura-Actividad , Transfección , Tubulina (Proteína)/química , Tubulina (Proteína)/efectos de los fármacosRESUMEN
Recent studies have suggested a correlation between increased expression of specific beta-tubulin isotypes and paclitaxel resistance in drug-selected cell lines. In an attempt to establish a causal link, we have transfected Chinese hamster ovary cells with cDNAs encoding epitope-tagged class I, II, and IVb beta-tubulins, as well as a class I beta-tubulin with a mutation previously characterized in a paclitaxel resistant mutant. To eliminate possible toxicity that might be associated with overexpression of non-native tubulin, each of the cDNAs was placed under the control of a tetracycline regulated promoter. All transfected cDNAs produced assembly competent tubulin whose synthesis could be turned off or on by the presence or absence of tetracycline. Production of betaI, betaII, or betaIVb tubulin had no effect on the sensitivity of the cells to paclitaxel, but production of the mutant betaI-tubulin conferred clear resistance to the drug. We conclude from these experiments that simple overexpression of class I, II, or IVb isoforms of beta-tubulin is insufficient to confer resistance to paclitaxel.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Paclitaxel/farmacología , Tubulina (Proteína)/clasificación , Tubulina (Proteína)/genética , Animales , Células CHO , Cricetinae , ADN Complementario/genética , Resistencia a Medicamentos/genética , Expresión Génica , Microscopía Fluorescente , Microtúbulos/metabolismo , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tetraciclina/farmacología , Transfección , Tubulina (Proteína)/metabolismoRESUMEN
The cardiac Na+-Ca2+ exchanger (NCX1) is the principal Ca2+ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and alpha-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from -184 to +172 is sufficient for cardiac expression and alpha-adrenergic stimulation. Mutational analysis revealed that both the CArG element at -80 and the GATA element at -50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the -172 E-box demonstrated that it was required for alpha-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (+103 to +134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the -172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B , Miocardio/metabolismo , Proteínas Represoras/fisiología , Intercambiador de Sodio-Calcio/genética , Factores de Transcripción/fisiología , Agonistas alfa-Adrenérgicos/farmacología , Animales , Animales Recién Nacidos , Secuencia de Bases , Proteínas de Ciclo Celular , Células Cultivadas , ADN , Corazón/efectos de los fármacos , Datos de Secuencia Molecular , Miocardio/citología , Fenilefrina/farmacología , Regiones Promotoras Genéticas , Ratas , RibonucleoproteínasRESUMEN
Increased microtubule density, through viscous loading of active myofilaments, causes contractile dysfunction of hypertrophied and failing pressure-overloaded myocardium, which is normalized by microtubule depolymerization. We have found this to be based on augmented tubulin synthesis and microtubule stability. We show here that increased tubulin synthesis is accounted for by marked transcriptional up-regulation of the beta1- and beta2-tubulin isoforms, that hypertrophic regulation of these genes recapitulates their developmental regulation, and that the greater proportion of beta1-tubulin protein may have a causative role in the microtubule stabilization found in cardiac hypertrophy.
Asunto(s)
Cardiomegalia/genética , Familia de Multigenes , Tubulina (Proteína)/genética , Secuencia de Aminoácidos , Animales , Cardiomegalia/patología , Gatos , Hipertrofia Ventricular Derecha/genética , Hipertrofia Ventricular Derecha/patología , Microtúbulos/ultraestructura , Datos de Secuencia Molecular , Contracción Miocárdica , Transcripción Genética , Tubulina (Proteína)/biosíntesis , Regulación hacia ArribaRESUMEN
The factors determining the pH dependence of the formation and decay of the O photointermediate of the bacteriorhodopsin (bR) photocycle were investigated in the wild-type (WT) pigment and in the mutants of Glu-194 and Glu-204, key residues of the proton release group (PRG) in bR. We have found that in the WT the rate constant of O --> bR transition decreases 30-fold upon decreasing the pH from 6 to 3 with a pKa of about 4.3. D2O slows the rise and decay of the O intermediate in the WT at pH 3.5 by a factor of 5.5. We suggest that the rate of the O --> bR transition (which reflects the rate of deprotonation of the primary proton acceptor Asp-85) at low pH is controlled by the deprotonation of the PRG. To test this hypothesis, we studied the E194D mutant. We show that the pKa of the PRG in the ground state of the E194D mutant, when Asp-85 is protonated, is increased by 1.2 pK units compared to that of the WT. We found a similar increase in the pKa of the rate constant of the O --> bR transition in E194D. This provides further evidence that the rate of the O --> bR transition is controlled by the PRG. In a further test, the E194Q mutation, which disables the PRG and slows proton release, almost completely eliminates the pH dependence of O decay at pHs below 6. A second phenomenon we investigated was that in the WT at neutral and alkaline pH the fraction of the O intermediate decreases with pKa 7.5. A similar pH dependence is observed in the mutants in which the PRG is disabled, E194Q and E204Q, suggesting that the decrease in the fraction of the O intermediate with pKa ca. 7.5 is not controlled by the PRG. We propose that the group with pKa 7.5 is Asp-96. The slowing of the reprotonation of Asp-96 at high pH is the cause of the decrease in the rate of the N --> O transition, leading to the decrease in the fraction of O.
Asunto(s)
Bacteriorodopsinas/química , Protones , Ácido Aspártico/química , Ácido Aspártico/genética , Azidas/química , Catálisis , Óxido de Deuterio/química , Ácido Glutámico/química , Ácido Glutámico/genética , Glutamina/química , Glutamina/genética , Halobacterium salinarum , Concentración de Iones de Hidrógeno , Cómputos Matemáticos , Modelos Químicos , Mutagénesis Sitio-Dirigida , Fotoquímica , VolumetríaRESUMEN
Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, alpha-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin left and right arrow Glu-tubulin) and then irreversible deglutamination (Glu-tubulin --> Delta2-tubulin), such that Glu- and Delta2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Delta2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Delta2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Delta2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.
Asunto(s)
Cardiomegalia/patología , Factor 1 Eucariótico de Iniciación , Microtúbulos/ultraestructura , Tubulina (Proteína)/metabolismo , Animales , Presión Sanguínea , Volumen Sanguíneo , Gatos , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genéticaRESUMEN
Substitution of glutamic acid-194, a residue on the extracellular surface of bacteriorhodopsin, with a cysteine inhibits the fast light-induced proton release that normally is coupled with the deprotonation of the Schiff base during the L to M transition. Proton release in this mutant occurs at the very end of the photocycle and coincides with deprotonation of the primary proton acceptor, Asp-85, during the O to bR transition. the E194C mutation also results in a slowing down of the photocycle by about 1 order of magnitude as compared to the wild type and produces a strong effect on the pH dependence of dark adaptation that is interpreted as a drastic reduction or elimination of the coupling between the primary proton acceptor Asp-85 and the proton release group. These data indicate that Glu-194 is a critical component of the proton release complex in bacteriorhodopsin.
Asunto(s)
Bacteriorodopsinas/metabolismo , Cisteína/genética , Ácido Glutámico/genética , Luz , Protones , Adaptación a la Oscuridad , Halobacterium , Concentración de Iones de Hidrógeno , Cinética , Mutación , Espectrofotometría AtómicaRESUMEN
We have examined light-induced currents in oriented membranes of the bacteriorhodopsin mutants R82K and R82Q. Our results suggest that two photocurrent components found in R82K, with 30 and 300 microseconds lifetimes, are due to the photocycle of the 13-cis rather than the all-trans form of the pigment. We investigated the pH dependence of these components and their correspondence to absorbance changes at 660 nm characteristic of photointermediates of the 13-cis cycle. The presence of a D2O effect suggests that the charge motions producing these photocurrents are related to proton or protonated amino acid movement within the molecule. The current amplitudes depend on the protonation states of at least two residues, D85 and (probably) E204. In R82Q, a 10 microseconds photocurrent is observed that also depends on the protonation state of D85 and is similar to the 30 microseconds current in R82K. We attempt to explain these currents in terms of a model for interacting residues in the extracellular half of the bacteriorhodopsin channel.
Asunto(s)
Bacteriorodopsinas/química , Luz , Adaptación Fisiológica , Bacteriorodopsinas/genética , Bacteriorodopsinas/efectos de la radiación , Óxido de Deuterio , Electroquímica , Mutación , FotoquímicaRESUMEN
The Na+-Ca2+ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5'-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5'-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (-2000 to -1250 base pairs (bp)) and one or more negative elements (-1250 to -250 bp), a minimum promoter (-250 to +200 bp) is sufficient for cardiac expression and alpha-adrenergic stimulation.
Asunto(s)
Proteínas Portadoras/genética , Miocardio/metabolismo , Regiones Promotoras Genéticas , Regulación hacia Arriba , Animales , Secuencia de Bases , Transporte Biológico , Encéfalo/metabolismo , Calcio/metabolismo , Cardiomegalia/metabolismo , Gatos , Clonación Molecular , Exones , Genes Reporteros , Biblioteca Genómica , Riñón/metabolismo , Células L , Ratones , Datos de Secuencia Molecular , Miocardio/citología , Proteínas Recombinantes de Fusión/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Sodio/metabolismo , Intercambiador de Sodio-Calcio , Distribución TisularRESUMEN
Given the central position of the focal adhesion complex, both physically in coupling integrins to the interstitium and biochemically in providing an upstream site for anabolic signal generation, we asked whether the recruitment of non-receptor tyrosine kinases to the cytoskeleton might be a mechanism whereby cellular loading could activate growth regulatory signals responsible for cardiac hypertrophy. Analysis revealed cytoskeletal association of c-Src, FAK, and beta3-integrin, but no Fyn, in the pressure-overloaded right ventricle. This association was seen as early as 4 h after right ventricular pressure overloading, increased through 48 h, and reverted to normal in 1 week. Cytoskeletal binding of non-receptor tyrosine kinases was synchronous with tyrosine phosphorylation of several cytoskeletal proteins, including c-Src. Examination of cytoskeleton-bound c-Src revealed that a significant portion of the tyrosine phosphorylation was not at the Tyr-527 site and therefore presumably was at the Tyr-416 site. Thus, these studies strongly suggest that non-receptor tyrosine kinases, in particular c-Src, may play a critical role in hypertrophic growth regulation by their association with cytoskeletal structures, possibly via load activation of integrin-mediated signaling.
Asunto(s)
Cardiomegalia/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Tirosina/metabolismo , Animales , Gatos , Proteínas del Citoesqueleto/metabolismo , Integrinas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismoRESUMEN
K129 is a residue located in the extracellular loop connecting transmembrane helices D and E of bacteriorhodopsin. Replacement of K129 with a histidine alters the pKa's of two key residues in the proton transport pathway, D85, and the proton release group (probably E204); the resulting pigment has properties that differ markedly from the wild type. 1) In the unphotolyzed state of the K129H mutant, the pKa of D85 is 5.1 +/- 0.1 in 150 mM KCl (compared to approximately 2.6 in the wild-type bacteriorhodopsin), whereas the unphotolyzed-state pKa of E204 decreases to 8.1 +/- 0.1 (from approximately 9.5 in the wild-type pigment). 2) The pKa of E204 in the M state is 7.0 +/- 0.1 in K129H, compared to approximately 5.8 in the wild-type pigment. 3) As a result of the change in the pKa of E204 in M, the order of light-induced proton release and uptake exhibits a dependence on pH in K129H differing from that of the wild type: at neutral pH and moderate salt concentrations (150 mM KCl), light-induced proton uptake precedes proton release, whereas it follows proton release at higher pH. This pumping behavior is similar to that seen in a related bacterial rhodopsin, archaerhodopsin-1, which has a histidine in the position analogous to K129. 4) At alkaline pH, a substantial fraction of all-trans K129H pigment (approximately 30%) undergoes a conversion into a shorter wavelength species, P480, with pKa approximately 8.1, close to the pKa of E204. 5) Guanidine hydrochloride lowers the pKa's of D85 and E204 in the ground state and the pKa of E204 in the M intermediate, and restores the normal order of proton release before uptake at neutral pH. 6) In the K129H mutant the coupling between D85 and E204 is weaker than in wild-type bacteriorhodopsin. In the unphotolyzed pigment, the change in the pKa's of either residue when the other changes its protonation state is only 1.5 units compared to 4.9 units in wild-type bacteriorhodopsin. In the M state of photolyzed K129H pigment, the corresponding change is 1 unit, compared to 3.7 units in the wild-type pigment. We suggest that K129 may be involved in stabilizing the hydrogen bonding network that couples E204 and D85. Substitution of K129 with a histidine residue causes structural changes that alter this coupling and affect the pKa's of E204 and D85.
Asunto(s)
Bacteriorodopsinas/metabolismo , Halobacterium/fisiología , Lisina/química , Protones , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Adaptación a la Oscuridad , Guanidina , Guanidinas/farmacología , Halobacterium/química , Halobacterium/genética , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Cinética , Luz , Mutagénesis Sitio-Dirigida , Mutación , Fotólisis , Bombas de Protones/fisiología , EspectrofotometríaRESUMEN
Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.
Asunto(s)
Arginina/metabolismo , Bacteriorodopsinas/metabolismo , Lisina/metabolismo , Arginina/genética , Bacteriorodopsinas/química , Bacteriorodopsinas/genética , Concentración de Iones de Hidrógeno , Cinética , Lisina/genética , Mutagénesis Sitio-Dirigida , ProtonesAsunto(s)
Calcio/metabolismo , Cardiomegalia/metabolismo , Proteínas Portadoras/genética , Expresión Génica , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ATPasas Transportadoras de Calcio/metabolismo , Datos de Secuencia Molecular , Miocardio/citología , Miocardio/metabolismo , Regulación hacia ArribaRESUMEN
In wild-type bacteriorhodopsin light-induced proton release occurs before uptake at neutral pH. In contrast, in mutants in which R82 is replaced by a neutral residue (as in R82A and R82Q), only a small fraction of the protons is released before proton uptake at neutral pH; the major fraction is released after uptake. In R82Q the relative amounts of the two types of proton release, "early" (preceding proton uptake) and "late" (following proton uptake), are pH dependent. The main conclusions are that 1) R82 is not the normal light-driven proton release group; early proton release can be observed in the R82Q mutant at higher pH values, suggesting that the proton release group has not been eliminated. 2) R82 affects the pKa of the proton release group both in the unphotolyzed state of the pigment and during the photocycle. In the wild type (in 150 mM salt) the pKa of this group decreases from approximately 9.5 in the unphotolyzed pigment to approximately 5.8 in the M intermediate, leading to early proton release at neutral pH. In the R82 mutants the respective values of pKa of the proton release group in the unphotolyzed pigment and in M are approximately 8 and 7.5 in R82Q (in 1 M salt) and approximately 8 and 6.5 in R82K (in 150 mM KCl). Thus in R82Q the pKa of the proton release group does not decrease enough in the photocycle to allow early proton release from this group at neutral pH. 3) Early proton release in R82Q can be detected as a photocurrent signal that is kinetically distinct from those photocurrents that are due to proton movements from the Schiff base to D85 during M formation and from D96 to the Schiff base during the M-->N transition. 4) In R82Q, at neutral pH, proton uptake from the medium occurs during the formation of O. The proton is released during the O-->bacteriorhodopsin transition, probably from D85 because the normal proton release group cannot deprotonate at this pH. 5) The time constant of early proton release is increased from 85 microseconds in the wild type to 1 ms in R82Q (in 150 mM salt). This can be directly attributed to the increase in the pKa of the proton release group and also explains the uncoupling of proton release from M formation. 6) In the E204Q mutant only late proton release is observed at both neutral and alkaline pH, consistent with the idea that E204 is the proton release group. The proton release is concurrent with the O-->bacteriorhodopsin transition, as in R82Q at neutral pH.