RESUMEN
PURPOSE: Accurate quantification of the intact proteins, antibodies or peptides and their impurities without interaction to silanols of HPLC column. METHODS: Hydroxypropyl ß Cyclodextrin (HPCD) is added in the mobile phase at different concentrations. Different commercial SEC-HPLC columns and biologics with a molecular weight ranging from 5.8 kDa to 150kDa were assessed with and without cyclodextrin. RESULTS: Addition of non-ionic sugars such as Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved peak performance such as theoretical plates, peak resolution, peak width, peak height, and improved quantification of aggregates in biologics such as antibodies Humira and Actemra, and peptides such as insulin. There is an increase in peak height, reduced retention time, increased plate and reduced peak width with increasing concentration of cyclodextrin studied. DISCUSSION: High ionic strength, basic amino acids such as arginine, organic solvents (with a concentration low enough not to precipitate protein), sodium perchlorate and ion pairing agents in the mobile phase used for separation of peptides, proteins and antibodies to prevent silanol interaction. These commonly used solutions are not always successful, as they not only interact with the biologic, but are sometimes, not compatible. The non-ionic cyclodextrin itself does not cause protein aggregation but prevents the nonspecific binding or interaction of protein itself and thereby allowing for improved resolution, and accurate quantification of aggregates in antibodies, and peptides. The data on the separation in presence of cyclodextrin in the mobile phase showed higher peak resolution, improved peak shape, accurate apparent molecular weight, improved efficiency, and less peak tailing for biological products. CONCLUSION: Hydroxypropyl ß Cyclodextrin in the mobile phase, resulted improved SEC-HPLC resolution, and quantitation of aggregates in biologics by preventing the interaction of biologics to silanol of the commercial SEC-HPLC columns.
Asunto(s)
2-Hidroxipropil-beta-Ciclodextrina/química , Cromatografía en Gel/métodos , Cromatografía Líquida de Alta Presión/métodos , Péptidos/análisis , Proteínas/análisis , Adalimumab/análisis , Animales , Anticuerpos Monoclonales Humanizados/análisis , Humanos , Inmunoglobulina G/análisis , Insulina/análisis , Agregado de Proteínas , Ratas , Silanos/químicaRESUMEN
The transient receptor potential vanilloid 1 (TRPV1) channel is an essential component of the cellular mechanism through which noxious stimuli evoke pain. Functional and structural characterizations of TRPV1 shed light on vanilloid activation, yet the mechanisms for temperature and proton gating remain largely unknown. Spectroscopic approaches are needed to understand the mechanisms by which TRPV1 translates diverse stimuli into channel opening. Here, we have engineered a minimal cysteine-less rat TRPV1 construct (eTRPV1) that can be stably purified and reconstituted for spectroscopic studies. Biophysical analyses of TRPV1 constructs reveal that the S5-pore helix loop influences protein stability and vanilloid and proton responses, but not thermal sensitivity. Cysteine mutants retain function and stability for double electron-electron resonance (DEER) and electron paramagnetic resonance (EPR) spectroscopies. DEER measurements in the closed state demonstrate that eTRPV1 reports distances in the extracellular vestibule, equivalent to those observed in the apo TRPV1 structure. EPR measurements show a distinct pattern of mobilities and spectral features, in detergent and liposomes, for residues at the pore domain that agree with their location in the TRPV1 structure. Our results set the stage for a systematic characterization of TRPV1 using spectroscopic approaches to reveal conformational changes compatible with thermal- and ligand-dependent gating.
Asunto(s)
Expresión Génica , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cisteína/química , Simulación de Dinámica Molecular , Mutación , Fosforilación , Conformación Proteica , Estabilidad Proteica , Protones , Ratas , Proteínas Recombinantes , Análisis Espectral , Canales Catiónicos TRPV/química , XenopusRESUMEN
Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca(2+)-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.
Asunto(s)
Canales de Calcio/metabolismo , Fosfatidilinositoles/química , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/química , Canales de Calcio/genética , Ciona intestinalis , Simulación por Computador , Células HEK293 , Humanos , Lípidos/química , Conformación Molecular , Simulación del Acoplamiento Molecular , Mutación , Oocitos/citología , Técnicas de Placa-Clamp , Fosfolipasas/química , Unión Proteica , Conformación Proteica , Canales Catiónicos TRPV/genética , Xenopus laevisRESUMEN
The transient receptor potential cation channel subfamily V member 1 (TRPV1) or vanilloid receptor 1 is a nonselective cation channel that is involved in the detection and transduction of nociceptive stimuli. Inflammation and nerve damage result in the up-regulation of TRPV1 transcription, and, therefore, modulators of TRPV1 channels are potentially useful in the treatment of inflammatory and neuropathic pain. Understanding the binding modes of known ligands would significantly contribute to the success of TRPV1 modulator drug design programs. The recent cryo-electron microscopy structure of TRPV1 only provides a coarse characterization of the location of capsaicin (CAPS) and resiniferatoxin (RTX). Herein, we use the information contained in the experimental electron density maps to accurately determine the binding mode of CAPS and RTX and experimentally validate the computational results by mutagenesis. On the basis of these results, we perform a detailed analysis of TRPV1-ligand interactions, characterizing the protein ligand contacts and the role of individual water molecules. Importantly, our results provide a rational explanation and suggestion of TRPV1 ligand modifications that should improve binding affinity.
Asunto(s)
Capsaicina/metabolismo , Diterpenos/metabolismo , Activación del Canal Iónico , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/metabolismo , Aminoácidos/metabolismo , Sitios de Unión , Capsaicina/química , Microscopía por Crioelectrón , Diterpenos/química , Electricidad , Ligandos , Simulación del Acoplamiento Molecular , Mutagénesis/genética , Proteínas Mutantes/química , Electricidad Estática , Canales Catiónicos TRPV/química , AguaRESUMEN
Recent high resolution structures of several pentameric ligand-gated ion channels have provided unprecedented details of their molecular architecture. However, the conformational dynamics and structural rearrangements that underlie gating and allosteric modulation remain poorly understood. We used a combination of electrophysiology, double electron-electron resonance (DEER) spectroscopy, and x-ray crystallography to investigate activation mechanisms in a novel functional chimera with the extracellular domain (ECD) of amine-gated Erwinia chrysanthemi ligand-gated ion channel, which is activated by primary amines, and the transmembrane domain of Gloeobacter violaceus ligand-gated ion channel, which is activated by protons. We found that the chimera was independently gated by primary amines and by protons. The crystal structure of the chimera in its resting state, at pH 7.0 and in the absence of primary amines, revealed a closed-pore conformation and an ECD that is twisted with respect to the transmembrane region. Amine- and pH-induced conformational changes measured by DEER spectroscopy showed that the chimera exhibits a dual mode of gating that preserves the distinct conformational changes of the parent channels. Collectively, our findings shed light on both conserved and divergent features of gating mechanisms in this class of channels, and will facilitate the design of better allosteric modulators.
Asunto(s)
Proteínas Bacterianas/química , Activación del Canal Iónico , Canales Iónicos Activados por Ligandos/química , Protones , Aminas/farmacología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Erwinia/química , Canales Iónicos Activados por Ligandos/agonistas , Canales Iónicos Activados por Ligandos/metabolismo , Datos de Secuencia Molecular , XenopusRESUMEN
Human interleukinâ 6 (IL-6) is a potent cytokine with immunomodulatory properties. As the influence of N-glycosylation on the inâ vivo activities of IL-6 could not be elucidated so far, a semisynthesis of homogeneous glycoforms of IL-6 was established by sequential native chemical ligation. The four cysteines of IL-6 are convenient for ligations and require only the short synthetic glycopeptide 43-48. The Cys-peptide 49-183 could be obtained recombinantly by cleavage of a SUMO tag. The fragment 1-42 was accessible by the simultaneous cleavage of two inteins, leading to the 1-42 thioester with the native N-terminus. Ligation and refolding studies showed that the inherently labile Asp-Pro bond 139-140 was detrimental for the sequential C- to N-terminal ligation. A reversed ligation sequence using glycopeptide hydrazides gave full-length IL-6 glycoproteins, which showed full bioactivity after efficient refolding and purification.
Asunto(s)
Interleucina-6/síntesis química , Isoformas de Proteínas/síntesis química , Cromatografía Líquida de Alta Presión , Humanos , Interleucina-6/química , Espectrometría de Masas , Isoformas de Proteínas/químicaRESUMEN
Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.
Asunto(s)
Cianobacterias/química , Activación del Canal Iónico/fisiología , Canales Iónicos Activados por Ligandos/química , Proteínas de la Membrana/química , Alcoholes/metabolismo , Regulación Alostérica , Cianobacterias/metabolismo , Canales Iónicos Activados por Ligandos/genética , Canales Iónicos Activados por Ligandos/metabolismo , Ligandos , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.
Asunto(s)
Proteínas Bacterianas/química , Cianobacterias , Canales Iónicos Activados por Ligandos/química , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Liposomas/química , Potenciales de la Membrana , Modelos Moleculares , Técnicas de Placa-Clamp , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Propiedades de SuperficieRESUMEN
Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.
Asunto(s)
Cianobacterias/metabolismo , Activación del Canal Iónico , Canales Iónicos/metabolismo , Concentración de Iones de Hidrógeno , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/química , Lidocaína/farmacología , Ligandos , ProteolípidosRESUMEN
Potassium (K(+))-channel gating is choreographed by a complex interplay between external stimuli, K(+) concentration and lipidic environment. We combined solid-state NMR and electrophysiological experiments on a chimeric KcsA-Kv1.3 channel to delineate K(+), pH and blocker effects on channel structure and function in a membrane setting. Our data show that pH-induced activation is correlated with protonation of glutamate residues at or near the activation gate. Moreover, K(+) and channel blockers distinctly affect the open probability of both the inactivation gate comprising the selectivity filter of the channel and the activation gate. The results indicate that the two gates are coupled and that effects of the permeant K(+) ion on the inactivation gate modulate activation-gate opening. Our data suggest a mechanism for controlling coordinated and sequential opening and closing of activation and inactivation gates in the K(+)-channel pore.
Asunto(s)
Canales de Potasio/metabolismo , Animales , Bacterias/metabolismo , Membrana Celular/metabolismo , Electrofisiología , Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Iones , Ligandos , Membrana Dobles de Lípidos/química , Espectroscopía de Resonancia Magnética , Ratones , Modelos Biológicos , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
Gating the ion-permeation pathway in K(+) channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K(+) channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K(+) channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K(+) channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K(+) channel in a membrane setting.
Asunto(s)
Canal de Potasio Kv1.3/química , Proteínas Bacterianas , Membrana Celular , Concentración de Iones de Hidrógeno , Canal de Potasio Kv1.3/fisiología , Liposomas , Espectroscopía de Resonancia Magnética , Micelas , Técnicas de Placa-Clamp , Porfirinas/farmacología , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/fisiología , Conformación ProteicaRESUMEN
Recently, a solid-state NMR study revealed that scorpion toxin binding leads to conformational changes in the selectivity filter of potassium channels. The exact nature of the conformational changes, however, remained elusive. We carried out all-atom molecular dynamics simulations that enabled us to cover the complete pathway of toxin approach and binding, and we validated our simulation results by using solid-state NMR data and electrophysiological measurements. Our structural model revealed a mechanism of cooperative toxin-induced conformational changes that accounts both for the signal changes observed in solid-state NMR and for the tight interaction between KcsA-Kv1.3 and Kaliotoxin. We show that this mechanism is structurally and functionally closely related to recovery from C-type inactivation. Furthermore, our simulations indicate heterogeneity in the binding modes of Kaliotoxin, which might serve to enhance its affinity for KcsA-Kv1.3 further by entropic stabilization.