RESUMO
Most animal cell types are classified as non-excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non-excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure-guided engineering. rEstus is most sensitive in the resting voltage range of non-excitable cells and offers a 3.6-fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non-excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real-time readout of electrical cell-cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non-invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non-excitable cell types.
Assuntos
Potenciais de Ação , Humanos , Células HEK293 , Potenciais de Ação/fisiologia , Potenciais da Membrana/fisiologia , Células MCF-7RESUMO
Selenomethionine (SeMet) randomly replaces methionine (Met) in protein translation. Because of strongly differing redox properties of SeMet and Met, SeMet mis-incorporation may have detrimental effects on protein function, possibly compromising the use of nutritional SeMet supplementation as an anti-oxidant. Studying the functional impact of SeMet in proteins on a cellular level is hampered by the lack of accurate and efficient methods for estimating the SeMet incorporation level in individual viable cells. Here we introduce and apply a method to measure the extent of SeMet incorporation in cellular proteins by utilizing a genetically encoded fluorescent methionine oxidation probe. Supplementation of SeMet in mammalian culture medium resulted in >84% incorporation of SeMet, and SeMet labeling as low as 5% was readily measured. Kinetics and extent of SeMet incorporation on the single-cell level under live-cell imaging conditions provided direct access to protein turn-over kinetics and SeMet redox properties in a cellular context. The method is furthermore suited for experiments utilizing high-throughput fluorescence microplate readers or fluorescence-activated cell sorting (FACS) analysis.
Assuntos
Antioxidantes , Selenometionina , Animais , Antioxidantes/metabolismo , Mamíferos/metabolismo , Metionina/metabolismo , Oxirredução , Proteínas/metabolismo , Selenometionina/metabolismoRESUMO
Heme, an iron-protoporphyrin IX complex, is a cofactor bound to various hemoproteins and supports a broad range of functions, such as electron transfer, oxygen transport, signal transduction, and drug metabolism. In recent years, there has been a growing recognition of heme as a non-genomic modulator of ion channel functions. Here, we show that intracellular free heme and hemin modulate human ether à go-go (hEAG1, Kv10.1) voltage-gated potassium channels. Application of hemin to the intracellular side potently inhibits Kv10.1 channels with an IC50 of about 4 nM under ambient and 63 nM under reducing conditions in a weakly voltage-dependent manner, favoring inhibition at resting potential. Functional studies on channel mutants and biochemical analysis of synthetic and recombinant channel fragments identified a heme-binding motif CxHx8H in the C-linker region of the Kv10.1 C terminus, with cysteine 541 and histidines 543 and 552 being important for hemin binding. Binding of hemin to the C linker may induce a conformational constraint that interferes with channel gating. Our results demonstrate that heme and hemin are endogenous modulators of Kv10.1 channels and could be exploited to modulate Kv10.1-mediated cellular functions.
Assuntos
Canais de Potássio Éter-A-Go-Go , Hemina , Humanos , Potenciais da MembranaRESUMO
Heme (Fe2+-protoporphyrin IX) is a well-known protein prosthetic group; however, heme and hemin (Fe3+-protoporphyrin IX) are also increasingly viewed as signaling molecules. Among the signaling targets are numerous ion channels, with intracellular-facing heme-binding sites modulated by heme and hemin in the sub-µM range. Much less is known about extracellular hemin, which is expected to be more abundant, in particular after hemolytic insults. Here we show that the human cardiac voltage-gated sodium channel hNaV1.5 is potently inhibited by extracellular hemin (IC 50 ≈ 80 nM), while heme, dimethylhemin, and protoporphyrin IX are ineffective. Hemin is selective for hNaV1.5 channels: hNaV1.2, hNaV1.4, hNaV1.7, and hNaV1.8 are insensitive to 1 µM hemin. Using domain chimeras of hNaV1.5 and rat rNaV1.2, domain II was identified as the critical determinant. Mutation N803G in the domain II S3/S4 linker largely diminished the impact of hemin on the cardiac channel. This profile is reminiscent of the interaction of some peptide voltage-sensor toxins with NaV channels. In line with a mechanism of select gating modifiers, the impact of hemin on NaV1.5 channels is reversely use dependent, compatible with an interaction of hemin and the voltage sensor of domain II. Extracellular hemin thus has potential to modulate the cardiac function.
Assuntos
Venenos de Aranha , Ratos , Humanos , Animais , Venenos de Aranha/química , Venenos de Aranha/farmacologia , Hemina/farmacologia , Sítios de Ligação , Ligação Proteica , Peptídeos/químicaRESUMO
Methionine oxidation is a reversible post-translational protein modification, affecting protein function, and implicated in aging and degenerative diseases. The detection of accumulating methionine oxidation in living cells or organisms, however, has not been achieved. Here we introduce a genetically encoded probe for methionine oxidation (GEPMO), based on the super-folder green fluorescent protein (sfGFP), as a specific, versatile, and integrating sensor for methionine oxidation. Placed at amino-acid position 147 in an otherwise methionine-less sfGFP, the oxidation of this specific methionine to methionine sulfoxide results in a ratiometric fluorescence change when excited with â¼400 and â¼470 nm light. The strength and homogeneity of the sensor expression is suited for live-cell imaging as well as fluorescence-activated cell sorting (FACS) experiments using standard laser wavelengths (405/488 nm). Expressed in mammalian cells and also in S. cerevisiae, the sensor protein faithfully reports on the status of methionine oxidation in an integrating manner. Variants targeted to membranes and the mitochondria provide subcellular resolution of methionine oxidation, e.g. reporting on site-specific oxidation by illumination of endogenous protoporphyrin IX.
Assuntos
Metionina , Saccharomyces cerevisiae , Animais , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mamíferos/metabolismo , Metionina/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Saccharomyces cerevisiae/metabolismoRESUMO
The cellular resting membrane potential (Vm) not only determines electrical responsiveness of excitable cells but also plays pivotal roles in non-excitable cells, mediating membrane transport, cell-cycle progression, and tumorigenesis. Studying these processes requires estimation of Vm, ideally over long periods of time. Here, we introduce two ratiometric genetically encoded Vm indicators, rArc and rASAP, and imaging and analysis procedures for measuring differences in average resting Vm between cell groups. We investigated the influence of ectopic expression of K+ channels and their disease-causing mutations involved in Andersen-Tawil (Kir2.1) and Temple-Baraitser (KV10.1) syndrome on median resting Vm of HEK293T cells. Real-time long-term monitoring of Vm changes allowed to estimate a 40-50 min latency from induction of transcription to functional Kir2.1 channels in HEK293T cells. The presented methodology is readily implemented with standard fluorescence microscopes and offers deeper insights into the role of the resting Vm in health and disease.
Assuntos
Expressão Ectópica do Gene/fisiologia , Potenciais da Membrana , Canais de Potássio Corretores do Fluxo de Internalização/genética , Síndrome de Andersen/genética , Células HEK293 , Hallux/anormalidades , Humanos , Deficiência Intelectual/genética , Unhas Malformadas/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Polegar/anormalidadesRESUMO
Recent research has revealed that ion channels and transporters can be important players in tumor development, progression, and therapy resistance in melanoma. For example, members of the ABC family were shown to support cancer stemness-like features in melanoma cells, while several members of the TRP channel family were reported to act as tumor suppressors.Also, many transporter proteins support tumor cell viability and thus suppress apoptosis induction by anticancer therapy. Due to the high number of ion channels and transporters and the resulting high complexity of the field, progress in understanding is often focused on single molecules and is in total rather slow. In this review, we aim at giving an overview about a broad subset of ion transporters, also illustrating some aspects of the field, which have not been addressed in detail in melanoma. In context with the other chapters in this special issue on "Transportome Malfunctions in the Cancer Spectrum," a comparison between melanoma and these tumors will be possible.
Assuntos
Melanoma , Humanos , Canais IônicosRESUMO
N-type inactivation of voltage-gated K+ channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary ß subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K+ channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvß1.1, Kvß1.2, and Kvß3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvß1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary ß subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.
Assuntos
Hemina/metabolismo , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Sítios de Ligação , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Células HEK293 , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Ligação Proteica , Ratos , XenopusRESUMO
Fast N-type inactivation of voltage-gated K+ (Kv) channels is important in fine-tuning of cellular excitability. To serve diverse cellular needs, N-type inactivation is regulated by numerous mechanisms. Here, we address how reactive sulfur species-the gaseous messenger H2S and polysulfides-affect N-type inactivation of the mammalian Kv channels Kv1.4 and Kv3.4. In both channels, the H2S donor NaHS slowed down inactivation with varying potency depending on the "aging" of NaHS solution. Polysulfides were > 1000 times more effective than NaHS with the potency increasing with the number of sulfur atoms (Na2S2 < Na2S3 < Na2S4). In Kv1.4, C13 in the N-terminal ball domain mediates the slowing of inactivation. In recombinant protein exposed to NaHS or Na2S4, a sulfur atom is incorporated at C13 in the protein. In Kv3.4, the N terminus harbors two cysteine residues (C6, C24), and C6 is of primary importance for channel regulation by H2S and polysulfides, with a minor contribution from C24. To fully eliminate the dependence of N-type inactivation on sulfhydration, both cysteine residues must be removed (C6S:C24S). Sulfhydration of a single cysteine residue in the ball-and-chain domain modulates the speed of inactivation but does not remove it entirely. In both Kv1.4 and Kv3.4, polysulfides affected the N-terminal cysteine residues when assayed in the whole-cell configuration; on-cell recordings confirmed that polysulfides also modulate K+ channel inactivation with undisturbed cytosol. These findings have collectively identified reactive sulfur species as potent modulators of N-type inactivation in mammalian Kv channels.
Assuntos
Sulfeto de Hidrogênio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Sulfetos/farmacologia , Linhagem Celular , Cisteína/metabolismo , Células HEK293 , Humanos , Transdução de Sinais/fisiologiaRESUMO
Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so-called CO-releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM-2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca2+- and voltage-dependent K+ channels (KCa1.1) and voltage-gated K+ channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM-2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM-2 has the propensity of forming Ru(CO)2 adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass-spectrometry analysis. For KCa1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM-2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM-2 targets. The strong CO-independent action of CORM-2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM-2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off-site effects similar to those reported here for CORM-2 are found for CORM-3, another ruthenium-based CORM, but are diminished when using iron-based CORM-S1 and absent for manganese-based CORM-EDE1.
Assuntos
Monóxido de Carbono/metabolismo , Compostos Organometálicos/farmacologia , Canais de Potássio/metabolismo , Células HEK293 , Histidina/metabolismo , Humanos , Concentração de Íons de HidrogênioRESUMO
Photonic experiments are of key importance in life sciences but light-induced side effects are serious confounding factors. Here we introduce roNaV2, an engineered voltage-gated Na+ channel harboring a selenocysteine in its inactivation motif, as a non-photonic, sensitive, gateable, and reversible sensor for membrane-delimited reactive species. roNaV2 allows for the assessment of chemical modification induced in fluorescence microscopy settings with high sensitivity and time resolution and it demonstrates the usefulness of ion channels as highly sensitive reporters of membrane processes.
Assuntos
Membrana Celular/metabolismo , Fótons , Espécies Reativas de Oxigênio/metabolismo , Selenocisteína/metabolismo , Canais de Sódio/metabolismo , Animais , Células HEK293 , Humanos , Luz , Oxirredução , Ratos , Fatores de TempoRESUMO
The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.
Assuntos
Calmodulina/metabolismo , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/química , Sítios de Ligação , Calmodulina/química , Cristalografia por Raios X , Canais de Potássio Éter-A-Go-Go/genética , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação ProteicaRESUMO
Stereospecific capillary electrophoresis-based methods for the analysis of methionine sulfoxide [Met(O)]-containing pentapeptides were developed in order to investigate the reduction of Met(O)-containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N-acetylated, C-terminally 2,4-dinitrophenyl (Dnp)-labeled pentapeptides ac-Lys-Phe-Met(O)-Lys-Lys-Dnp, ac-Lys-Asp-Met(O)-Asn-Lys-Dnp and ac-Lys-Asn-Met(O)-Asp-Lys-Dnp was achieved in 50 mM Tris-HCl buffers containing sulfated ß-CD in fused-silica capillaries, while the diastereomer separation of ac-Lys-Asp-Met(O)-Asp-Lys-Dnp was achieved by sulfated ß-CD-mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild-type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild-type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac-Lys-Asn-Met(O)-Asp-Lys-Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac-Lys-Asp-Met(O)-Asn-Lys-Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met-S-(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.
Assuntos
Aspergillus nidulans/enzimologia , Eletroforese Capilar/métodos , Metionina Sulfóxido Redutases/metabolismo , Mutação , Oligopeptídeos/análise , Limite de Detecção , Metionina Sulfóxido Redutases/genética , Reprodutibilidade dos Testes , Especificidade por SubstratoRESUMO
Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 µM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.
Assuntos
Potenciais de Ação , Gânglios Espinais/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.8/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/citologia , Camundongos , Neurônios/fisiologiaRESUMO
A-type K(+) channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K(+) channel's α or ß subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvß1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca(2+) concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca(2+) dependence is gained by binding of calmodulin to the "chain" segment of Kvß1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca(2+)/calmodulin does not interfere with the ß subunit's enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvß1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.
Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , Canal de Potássio Kv1.1/metabolismo , Células Musculares/metabolismo , Neurônios/metabolismo , Animais , Citosol/metabolismo , Humanos , Canal de Potássio Kv1.2/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oócitos/metabolismo , Porosidade , Ratos , XenopusRESUMO
Rheumatoid arthritis synovial fibroblasts (RA-SFs) show an aggressive phenotype and support joint inflammation and tissue destruction. New druggable targets in RA-SFs would therefore be of high therapeutic interest. The present study shows that the intermediate-conductance, calcium-activated potassium channel KCa3.1 (KCNN4) is expressed at the mRNA and protein level in RA-SFs, is functionally active, and has a regulatory impact on cell proliferation and secretion of pro-inflammatory and pro-destructive mediators. Whole-cell patch-clamp recordings identified KCa3.1 as the dominant potassium channel in the physiologically relevant membrane voltage range below 0 mV. Stimulation with transforming growth factor ß1 (TGF-ß1) significantly increased transcription, translation, and channel function of KCa3.1. Inhibition of KCa3.1 by the selective, pore-blocking inhibitor TRAM-34, (and, in part, by siRNA) significantly reduced cell proliferation, as well as expression and secretion of pro-inflammatory factors (IL-6, IL-8, and MCP1) and the tissue-destructive protease MMP3. These effects were observed in non-stimulated and/or TGF-ß1-stimulated RA-SFs. Since small molecule-based interference with KCa3.1 is principally well tolerated in clinical settings, further evaluation of channel blockers in models of rheumatoid arthritis may be a promising approach to identify new pharmacological targets and develop new therapeutic strategies for this debilitating disease.
Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Membrana Sinovial/química , Artrite Reumatoide/patologia , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Metaloproteases/genética , Metaloproteases/metabolismo , Pirazóis/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membrana Sinovial/citologiaRESUMO
A dual-selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15-crown-5, 18-crown-6, Kryptofix® 21 and Kryptofix® 22 and ß-cyclodextrin, carboxymethyl-ß-cyclodextrin, and sulfated ß-cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 µm id fused-silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated ß-cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza-crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide-crown ether-cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.
Assuntos
Eletroforese Capilar/métodos , Metionina/análogos & derivados , Peptídeos/química , Éteres de Coroa/química , Ciclodextrinas/química , Eletroforese Capilar/instrumentação , Metionina/análise , Estrutura Molecular , EstereoisomerismoRESUMO
A capillary electrophoresis method has been developed and validated to evaluate the stereospecific activity of recombinant human methionine sulfoxide reductase enzymes employing the C-terminally dinitrophenyl-labeled N-acetylated pentapeptide ac-KIFM(O)K-Dnp as substrate (M(O)=methionine sulfoxide). The separation of the ac-KIFM(O)K-Dnp diastereomers and the reduced peptide ac-KIFMK-Dnp was optimized using experimental design with regard to the buffer pH, buffer concentration, sulfated ß-cyclodextrin and 15-crown-5 concentration as well as capillary temperature and separation voltage. A fractional factorial response IV design was employed for the identification of the significant factors and a five-level circumscribed central composite design for the final method optimization. Resolution of the peptide diastereomers as well as analyte migration time served as responses in both designs. The resulting optimized conditions included 50mM Tris buffer, pH 7.85, containing 5mM 15-crown-5 and 14.3mg/mL sulfated ß-cyclodextrin, at an applied voltage of 25kV and a capillary temperature of 21.5°C. The assay was subsequently applied to the determination of the stereospecificity of recombinant human methionine sulfoxide reductases A and B2. The Michaelis-Menten kinetic data were determined. The pentapeptide proved to be a good substrate for both enzymes. Furthermore, the first separation of methionine sulfoxide peptide diastereomers is reported.
Assuntos
Eletroforese Capilar/métodos , Metionina Sulfóxido Redutases/química , Peptídeos/química , Eletroforese Capilar/instrumentação , Humanos , Concentração de Íons de Hidrogênio , Cinética , Metionina/análogos & derivados , Metionina/química , Estereoisomerismo , Especificidade por SubstratoRESUMO
An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of L-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-L-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-L-methionine sulfoxide and the product Fmoc-L-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-L-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-L-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis-Menten constant, K m, and the maximum velocity, v max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.
Assuntos
Eletroforese Capilar/métodos , Ensaios Enzimáticos/métodos , Metionina Sulfóxido Redutases/metabolismo , Metionina/análogos & derivados , Fatores de Transcrição/metabolismo , Humanos , Cinética , Metionina/química , Metionina/metabolismo , Proteínas dos Microfilamentos , Modelos Moleculares , EstereoisomerismoRESUMO
In molecular interaction studies based on surface plasmon resonance (SPR) measurements, the ligand is often immobilized in a thin carboxydextran gel matrix. Here we investigated the influence of the charged gel on the results of such SPR measurements. At physiological ionic strength, analytes with a net charge of more than about 5 are considerably enriched or depleted due to the Donnan potential under commonly applied experimental conditions. Below physiological ionic strength, enrichment was found to be even stronger than predicted by Donnan theory. The influence of the gel matrix on the apparent binding is prevented in competition experiments, in which SPR measurements are only used to discriminate between free and complexed analyte while the interaction between analyte and ligand is studied in solution. However, if the analyte-ligand interaction is very fast, thermodynamic equilibrium is disturbed near the interface where free analyte binds to the immobilized ligand due to mass transport limitation. Consequently, the soluble analyte-ligand complex dissociates, which results in an overestimation of free analyte. In experiments of calmodulin binding to fragments of the KCNH1 ion channel protein this mass-transport-induced dissociation led to a systematic underestimation of the affinity. We conclude that the insufficient discrimination between the true analyte-ligand binding and the complex interactions of the analyte with the gel phase may result in systematic errors. The theoretical framework for recognizing and avoiding such errors is provided.