RESUMEN
Chemical cross-linking combined with mass spectrometry provides a powerful method for identifying protein-protein interactions and probing the structure of protein complexes. A number of strategies have been reported that take advantage of the high sensitivity and high resolution of modern mass spectrometers. Approaches typically include synthesis of novel cross-linking compounds, and/or isotopic labeling of the cross-linking reagent and/or protein, and label-free methods. We report Xlink-Identifier, a comprehensive data analysis platform that has been developed to support label-free analyses. It can identify interpeptide, intrapeptide, and deadend cross-links as well as underivatized peptides. The software streamlines data preprocessing, peptide scoring, and visualization and provides an overall data analysis strategy for studying protein-protein interactions and protein structure using mass spectrometry. The software has been evaluated using a custom synthesized cross-linking reagent that features an enrichment tag. Xlink-Identifier offers the potential to perform large-scale identifications of protein-protein interactions using tandem mass spectrometry.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Péptidos/química , Programas Informáticos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Estructura Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Espectrometría de Masas en Tándem/instrumentación , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
Chemical cross-linking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of cross-linked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact cross-linkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under conditions where the native complex structure is stable. In this study, we present a novel compact cross-linker that contains two distinct features: (1) an alkyne tag and (2) a small molecule detection tag (NO(2)) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the cross-linked peptides after proteolytic cleavage and coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO(2) moiety provides a secondary means of detecting cross-linked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this cross-linker, which we termed CLIP (click-enabled linker for interacting proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for cross-linked ubiquitin in highly complex E. coli cell lysates. Sequential collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and electron transfer dissociation (ETD)-MS/MS of intercross-linked peptides (two peptides connected with a cross-linker) are also demonstrated for improved automated identification of cross-linked peptides.
Asunto(s)
Reactivos de Enlaces Cruzados/química , Péptidos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodos , Ubiquitina/química , Alquinos/química , Secuencia de Aminoácidos , Sitios de Unión , Datos de Secuencia Molecular , Péptidos/química , Unión Proteica , Ubiquitina/metabolismoRESUMEN
Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein-protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic-sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification.
Asunto(s)
Marcadores de Afinidad/química , Reactivos de Enlaces Cruzados/química , Colorantes Fluorescentes/química , Proteínas/química , Marcadores de Afinidad/síntesis química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calmodulina/química , Calmodulina/metabolismo , Línea Celular Tumoral , Reactivos de Enlaces Cruzados/síntesis química , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Colorantes Fluorescentes/síntesis química , Ratones , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/metabolismo , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/metabolismoRESUMEN
Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT(2)). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT(2). Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 A increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K(d) = 4.8 +/- 0.4 microM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.
Asunto(s)
ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Calcio/farmacología , Nucleótidos/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , ATPasas Transportadoras de Calcio/biosíntesis , ATPasas Transportadoras de Calcio/genética , Activación Enzimática/efectos de los fármacos , Transferencia Resonante de Energía de Fluorescencia , Expresión Génica , Modelos Moleculares , Movimiento , Estructura Terciaria de Proteína , RatasRESUMEN
The structural coupling between opposing domains of CaM was investigated using the conformationally sensitive biarsenical probe 4,5-bis(1,3,2-dithioarsolan-2-yl)resorufin (ReAsH), which upon binding to an engineered tetracysteine motif near the end of helix A (Thr-5 to Phe-19) becomes highly fluorescent. Changes in conformation and dynamics are reflective of the native CaM structure, as there is no change in the (1)H- (15)N HSQC NMR spectrum in comparison to wild-type CaM. We find evidence of a conformational intermediate associated with CaM activation, where calcium occupancy of sites in the amino-terminal and carboxyl-terminal lobes of CaM differentially affect the fluorescence intensity of bound ReAsH. Insight into the structure of the conformational intermediate is possible from a consideration of calcium-dependent changes in rates of ReAsH binding and helix A mobility, which respectively distinguish secondary structural changes associated with helix A stabilization from the tertiary structural reorganization of the amino-terminal lobe of CaM necessary for high-affinity binding to target proteins. Helix A stabilization is associated with calcium occupancy of sites in the carboxyl-terminal lobe ( K d = 0.36 +/- 0.04 microM), which results in a reduction in the rate of ReAsH binding from 4900 M (-1) s (-1) to 370 M (-1) s (-1). In comparison, tertiary structural changes involving helix A and other structural elements in the amino-terminal lobe require calcium occupancy of amino-terminal sites (K d = 18 +/- 3 microM). Observed secondary and tertiary structural changes involving helix A in response to the sequential calcium occupancy of carboxyl- and amino-terminal lobe calcium binding sites suggest an important involvement of helix A in mediating the structural coupling between the opposing domains of CaM. These results are discussed in terms of a model in which carboxyl-terminal lobe calcium activation induces secondary structural changes within the interdomain linker that release helix A, thereby facilitating the formation of calcium binding sites in the amino-terminal lobe and linked tertiary structural rearrangements to form a high-affinity binding cleft that can associate with target proteins.
Asunto(s)
Calcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Arsenicales/química , Sitios de Unión , Modelos Moleculares , Conformación Proteica , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.
Asunto(s)
Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Calmodulina/metabolismo , Catálisis , Clonación Molecular , Secuencia Conservada , Fusión Génica , Metionina/análogos & derivados , Metionina/metabolismo , Metionina Sulfóxido Reductasas , Datos de Secuencia Molecular , Alineación de Secuencia , Shewanella/enzimología , Shewanella/genética , EstereoisomerismoRESUMEN
Biarsenical multiuse affinity probes (MAPs) complexed with ethanedithiol (EDT) permit the selective cellular labeling of proteins engineered with tetracysteine motifs, but are limited by the availability of a single binding motif (i.e., CCPGCC or PG tag) that prevents the differential labeling of coexpressed proteins. To overcome this problem, we have used a high-throughput peptide screen to identify an alternate binding motif (i.e., CCKACC or KA tag), which has a similar brightness to the classical sequence upon MAP binding, but displays altered rates and affinities of association that permit the differential labeling of these peptide sequences by the red probe 4,5-bis(1,3,2-dithiarsolan-2-yl)-resorufin (ReAsH-EDT2) or its green cognate 4',5'-bis(1,3,2-dithoarsolan-2-yl)fluorescein (FLAsH-EDT2). The utility of this labeling strategy was demonstrated following the expression of PG- and KA-tagged subunits of RNA polymerase in E. coli. Specific labeling of two subunits of RNA polymerase in cellular lysates was achieved, whereby ReAsH-EDT2 is shown to selectively label the PG-tag on RNA polymerase alpha-subunit prior to the labeling of the KA-tag sequence of the beta-subunit of RNA polymerase with FlAsH-EDT2. These results demonstrate the ability to selectively label multiple individual proteins with orthogonal sequence tags in complex cellular lystates with spectroscopically distinct MAPs, and indicate the absolute specificity of ReAsH to target expressed proteins with essentially no nonspecific binding interactions.
Asunto(s)
Arsenicales/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Compuestos Organometálicos/metabolismo , Oxazinas/metabolismo , Péptidos/metabolismo , Secuencias de Aminoácidos , Clonación Molecular , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Análisis por Matrices de Proteínas , Unión ProteicaRESUMEN
Phospholamban (PLB) associates with the Ca(2+)-ATPase in sarcoplasmic reticulum (SR) membranes to permit the modulation of contraction in response to beta-adrenergic signaling. To understand how coordinated changes in the abundance and intracellular trafficking of PLB and the Ca(2+)-ATPase contribute to the maturation of functional muscle, we measured changes in abundance, location, and turnover of endogenous and tagged proteins in myoblasts and during their differentiation. We found that PLB is constitutively expressed in both myoblasts and differentiated myotubes, whereas abundance increases of the Ca(2+)-ATPase coincide with the formation of differentiated myotubes. We observed that PLB is primarily present in highly mobile vesicular structures outside the endoplasmic reticulum, irrespective of the expression of the Ca(2+)-ATPase, indicating that PLB targeting is regulated through vesicle trafficking. Moreover, using pulse-chase methods, we observed that in myoblasts, PLB is trafficked through directed transport through the Golgi to the plasma membrane before endosome-mediated internalization. The observed trafficking of PLB to the plasma membrane suggests an important role for PLB during muscle differentiation, which is distinct from its previously recognized role in the regulation of the Ca(2+)-ATPase.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular , Células Musculares/citología , Células Musculares/metabolismo , Retículo Sarcoplasmático/metabolismo , Animales , Biomarcadores , Proteínas de Unión al Calcio/genética , Línea Celular , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes , Ratones , Transporte de Proteínas , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Directed binding of RNA polymerase to distinct promoter elements controls transcription and promotes adaptive responses to changing environmental conditions. To identify proteins that modulate transcription, we have expressed a tagged alpha-subunit of RNA polymerase in Shewanella oneidensis under controlled growth conditions, isolated the protein complex using newly developed multiuse affinity probes, and used LC-MS/MS to identify proteins in the complex. Complementary fluorescence correlation spectroscopy measurements were used to determine the average size of the RNA polymerase complex in cellular lysates. We find that RNA polymerase exists as a large supramolecular complex with an apparent mass in excess of 1.4 MDa, whose protein composition substantially changes in response to growth conditions. Enzymes that copurify with RNA polymerase include those associated with tRNA processing, nucleotide metabolism, and energy biosynthesis, which we propose to be necessary for optimal transcriptional rates.
Asunto(s)
Aerobiosis/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Sustancias Macromoleculares/metabolismo , Shewanella/enzimología , Transcripción Genética , Adenosina Trifosfato/metabolismo , Anaerobiosis , Cromatografía de Afinidad , Cromatografía Liquida , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/aislamiento & purificación , Peso Molecular , Shewanella/crecimiento & desarrollo , Espectrometría de Fluorescencia , Espectrometría de Masas en TándemRESUMEN
The purified outer membrane bacterial protein OmcA binds densely to the surface of hematite (Fe2O3), permitting direct electron transfer to this solid mineral to reduce Fe (III) with an electron flux of about 1013 electrons /cm2/s. In the presence of hematite, there is a substantial increase in the amplitude of internal protein motions that correlate with metal reduction. Binding is highly favorable, with a partition coefficient of approximately 2 x 105 (DeltaGo' = -28 kJ/mol), where approximately 1014 OmcA proteins bind per cm2 to the solid metal surface, indicating the utility of using purified OmcA in the construction of a biofuel cell.
Asunto(s)
Proteínas Bacterianas/metabolismo , Citocromos c/metabolismo , Proteínas de la Membrana/metabolismo , Metales/metabolismo , Shewanella/enzimología , Proteínas Bacterianas/química , Citocromos c/química , Proteínas de la Membrana/química , Unión Proteica , Espectrometría de FluorescenciaRESUMEN
Shewanella oneidensis MR-1 is a facultatively anaerobic bacterium capable of using soluble and insoluble forms of manganese [Mn(III/IV)] and iron [Fe(III)] as terminal electron acceptors during anaerobic respiration. To assess the structural association of two outer membrane-associated c-type decaheme cytochromes (i.e., OmcA [SO1779] and MtrC [SO1778]) and their ability to reduce soluble Fe(III)-nitrilotriacetic acid (NTA), we expressed these proteins with a C-terminal tag in wild-type S. oneidensis and a mutant deficient in these genes (i.e., Delta omcA mtrC). Endogenous MtrC copurified with tagged OmcA in wild-type Shewanella, suggesting a direct association. To further evaluate their possible interaction, both proteins were purified to near homogeneity following the independent expression of OmcA and MtrC in the Delta omcA mtrC mutant. Each purified cytochrome was confirmed to contain 10 hemes and exhibited Fe(III)-NTA reductase activity. To measure binding, MtrC was labeled with the multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (1,2-ethanedithiol)2, which specifically associates with a tetracysteine motif engineered at the C terminus of MtrC. Upon titration with OmcA, there was a marked increase in fluorescence polarization indicating the formation of a high-affinity protein complex (Kd < 500 nM) between MtrC and OmcA whose binding was sensitive to changes in ionic strength. Following association, the OmcA-MtrC complex was observed to have enhanced Fe(III)-NTA reductase specific activity relative to either protein alone, demonstrating that OmcA and MtrC can interact directly with each other to form a stable complex that is consistent with their role in the electron transport pathway of S. oneidensis MR-1.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Grupo Citocromo c/metabolismo , Oxidorreductasas/metabolismo , Shewanella/enzimología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Grupo Citocromo c/química , Grupo Citocromo c/aislamiento & purificación , Transporte de Electrón , Compuestos Férricos/metabolismo , Hemo , Familia de Multigenes , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
CrAsH is a tetracysteine-binding probe which has improved properties in terms of signal-to-noise ratio and pH dependence of fluorescence compared to the parent compound.
Asunto(s)
Albúminas/química , Colorantes Fluorescentes/química , Compuestos Organometálicos/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Péptidos/químicaRESUMEN
Fluorophore-assisted light inactivation (FALI) permits the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding target protein sensitivity. To permit the rational inactivation of targeted proteins, we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins, regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between approximately 7 and 40 A. In addition, we observed a loss of histidines, as well as zero-length cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces.
Asunto(s)
Calmodulina/metabolismo , Fluoresceínas/farmacología , Colorantes Fluorescentes/farmacología , Luz , Metionina/metabolismo , Compuestos Organometálicos/farmacología , Oxidación-Reducción , Oxígeno Singlete/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Cisteína/genética , Cisteína/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Histidina/química , Histidina/metabolismo , Cinética , Espectrometría de Masas , Ratones , Datos de Secuencia Molecular , Quinasa de Cadena Ligera de Miosina/metabolismo , Compuestos Organometálicos/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Estructura Terciaria de Proteína , Conejos , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Oxígeno Singlete/metabolismo , Tripsina/metabolismoRESUMEN
Stabilization of the plasma membrane Ca-ATPase (PMCA) in an inactive conformation upon oxidation of multiple methionines in the calcium regulatory protein calmodulin (CaM) is part of an adaptive cellular response to minimize ATP utilization and the generation of reactive oxygen species (ROS) under conditions of oxidative stress. To differentiate oxidant-induced structural changes that selectively modify the amino-terminal domain of CaM from those that modulate the conformational coupling between the opposing domains, we have engineered a tetracysteine binding motif within helix A in the amino-terminal domain of calmodulin (CaM) that permits the selective and rigid attachment of the conformationally sensitive fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein-(1,2-ethanedithiol)(2) (FlAsH-EDT(2)). The position of the FlAsH label in the amino-terminal domain provides a signal for monitoring its binding to the CaM-binding sequence of the PMCA. Following methionine oxidation, there is an enhanced binding affinity between the amino-terminal domain and the CaM-binding sequence of the PMCA. To identify oxidant-induced structural changes, we used frequency domain fluorescence anisotropy measurements to assess the structural coupling between helix A and the amino- and carboxyl-terminal domains of CaM. Helix A undergoes large amplitude motions in apo-CaM; following calcium activation, helix A is immobilized as part of a conformational switch that couples the opposing domains of CaM to stabilize the high-affinity binding cleft associated with target protein binding. Methionine oxidation disrupts the structural coupling between opposing globular domains of CaM, without affecting the calcium-dependent immobilization of helix A associated with activation of the amino-terminal domain to promote high-affinity binding to target proteins. We suggest that this selective disruption of the structural linkage between the opposing globular domains of CaM relieves steric constraints associated with high-affinity target binding, permitting the formation of new contact interactions between the amino-terminal domain and the CaM-binding sequence that stabilizes the PMCA in an inhibited conformation.
Asunto(s)
ATPasas Transportadoras de Calcio/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/química , Calmodulina/química , Calmodulina/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Secuencia de Aminoácidos , ATPasas Transportadoras de Calcio/metabolismo , Calmodulina/genética , Membrana Celular/enzimología , Electroforesis en Gel de Poliacrilamida , Ensayo de Cambio de Movilidad Electroforética , Fluoresceínas/química , Fluoresceínas/metabolismo , Polarización de Fluorescencia/métodos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Compuestos Organometálicos/química , Compuestos Organometálicos/metabolismo , Oxidación-Reducción , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solventes , Espectrometría de FluorescenciaRESUMEN
Calcium-dependent changes in the internal dynamics and average structures of the opposing globular domains of calmodulin (CaM), as well as their relative spatial arrangement, contribute to the productive association between CaM and a range of different target proteins, affecting their functional activation. To identify dynamic structural changes involving individual alpha-helical elements and their modulation by calcium activation, we have used site-directed mutagenesis to engineer a tetracysteine binding motif within helix A near the amino terminus of calmodulin (CaM), permitting the selective and rigid attachment of the fluorescent probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH) with full retention of function. The rigid tetracoordinate linkage of FlAsH to CaM, in conjunction with frequency domain fluorescence anisotropy measurements, allows assessment of dynamic changes associated with calcium activation without interference from independent probe motion. Taking advantage of the large fluorescence enhancement associated with binding of FlAsH to CaM, we determined rates of binding of FlAsH to apo-CaM and calcium-activated CaM to be 2800 +/- 80 and 310 +/- 10 M(-)(1) s(-)(1), respectively. There is no difference in the solvent accessibility of the bound FlAsH irrespective of calcium binding to CaM. Thus, given that FlAsH selectively labels disordered structures, the large difference in rates of FlAsH binding indicates that calcium binding stabilizes helix A. Frequency domain anisotropy measurements of bound FlAsH indicate that prior to calcium activation, helix A undergoes large amplitude nanosecond motions. Following calcium activation, helix A becomes immobile, and structurally coupled to the overall rotation of CaM. We discuss these results in the context of a model that suggests stabilization of helix A relative to other domain elements in the CaM structure is critical to defining high-affinity binding clefts, and in promoting specific and ordered binding of the opposing lobes of CaM to target proteins.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Secuencia de Bases , Calmodulina/química , Calmodulina/genética , Cartilla de ADN , Fluoresceínas , Polarización de Fluorescencia , Colorantes Fluorescentes , Mutagénesis Sitio-Dirigida , Compuestos Organometálicos , Conformación ProteicaRESUMEN
Antihydrophobic cosolvents such as ethanol increase the solubility of hydrophobic molecules in water, and they also affect the rates of reactions involving hydrophobic surfaces. In simple reactions of hydrocarbons, such as the Diels-Alder dimerization of 1,3-cyclopentadiene, the rate and solubility data directly reflect the geometry of the transition state, in which some hydrophobic surface becomes hidden. In reactions involving polar groups, such as alkylations of phenoxide ions or S(N)1 ionizations of alkyl halides, cosolvents in water can have other effects as well. However, solvation of hydrophobic surfaces is still important. By the use of structure-reactivity relationships, and comparing the effects of ethanol and DMSO as solvents, it has been possible to sort out these effects. The conclusions are reinforced by an ab initio computer model for hydrophobic solvation. The result is a sensible transition state for phenoxide ion as a nucleophile, using its oxygen n electrons to avoid loss of conjugation. The geometry of alkylation of aniline is very different, involving packing (stacking) of the aniline ring onto the phenyl ring of a benzyl group in the benzylation reaction. The alkylation of phenoxide ions by benzylic chlorides can occur both at the phenoxide oxygen and on ortho and para positions of the ring. Carbon alkylation occurs in water, but not in nonpolar organic solvents, and it is observed only when the phenoxide has at least one methyl substituent ortho, meta, or para. The effects of phenol substituents and of antihydrophobic cosolvents on the rates of the competing alkylation processes indicate that in water the carbon alkylation involves a transition state with hydrophobic packing of the benzyl group onto the phenol ring. The results also support our conclusion that oxygen alkylation uses the n electrons of the phenoxide oxygen as the nucleophile and does not have hydrophobic overlap in the transition state. The mechanisms and explanations for competing oxygen and carbon alkylations differ from previous proposals by others.