RESUMEN
For irreversible denaturation transitions such as those exhibited by monoclonal antibodies, differential scanning calorimetry provides the denaturation temperature, Tm, the rate of denaturation at Tm, and the activation energy at Tm. These three quantities are essential but not sufficient for an accurate extrapolation of the rate of denaturation to temperatures of 25 °C and below. We have observed that the activation energy is not constant but temperature dependent due to the existence of an activation heat capacity, Cp,a. It is shown in this paper that a model that incorporates Cp,a is able to account for previous observations like, for example, that increasing the Tm does not always improve the stability at low temperatures; that some antibodies exhibit lower stabilities at 5 °C than at 25 °C; or that low temperature stabilities do not follow the rank order derived from Tm values. Most importantly, the activation heat capacity model is able to reproduce time dependent stabilities measured by size exclusion chromatography at low temperatures.
Asunto(s)
Anticuerpos Monoclonales , Rastreo Diferencial de Calorimetría , Desnaturalización Proteica , Anticuerpos Monoclonales/química , Frío , Temperatura , Estabilidad Proteica , TermodinámicaRESUMEN
Voltage-gated sodium channels, NaVs, are responsible for the rapid rise of action potentials in excitable tissues. NaV channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-NaV nanobodies (Nbs), Nb17 and Nb82, that recognize the NaV1.4 (skeletal muscle) and NaV1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of NaV1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human NaV isoform has been challenging because they usually show undesired crossreactivity for different NaV isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNaV1.4 or CTNaV1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNaV1.4 and CTNaV1.5 with high affinity (KD â¼ 40-60 nM). In addition, as proof of concept, we show that Nb82 could detect NaV1.4 and NaV1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo NaV1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-NaV reagents to capture NaVs from cell lysates and as molecular visualization agents for NaVs.
Asunto(s)
Anticuerpos de Dominio Único , Canales de Sodio Activados por Voltaje , Animales , Células Cultivadas , Escherichia coli/genética , Humanos , Síndrome de QT Prolongado/metabolismo , Mamíferos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Canales de Sodio Activados por Voltaje/genética , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
There have been numerous studies of the temperature denaturation of monoclonal antibodies (mAbs) using differential scanning calorimetry (DSC). In general, mAbs are characterized by complex temperature denaturation transitions in which the various domains (CH2, CH3, Fab) give rise to different peaks in the heat capacity function. The complexity and overall irreversibility of the temperature denaturation transition is well known and has limited the number of publications with an in-depth analysis of the data. Here we report that the temperature denaturation of the CH2 domain is reversible and only becomes irreversible after denaturation of the Fab domain, which is intrinsically irreversible. For these studies we have used the HIV neutralizing monoclonal antibody 17b. To account for the experimental heat capacity function, a mixed denaturation model that combines multiple reversible and irreversible transitions has been developed. This model accounts well for the DSC data and for the pH dependence of the heat capacity function of 17b and other monoclonal antibodies for which data is available in the literature. It is expected that a more detailed analysis of the stability of monoclonal antibodies will contribute to the development of better approaches to understand and optimize the structural viability of these therapeutic macromolecules.
Asunto(s)
Anticuerpos Monoclonales/química , Rastreo Diferencial de Calorimetría/métodos , Desnaturalización Proteica , Temperatura , Concentración de Iones de Hidrógeno , TermodinámicaRESUMEN
Many proteins are intrinsically disordered or contain one or more disordered domains. These domains can participate in binding interactions with other proteins or small ligands. Binding to intrinsically disordered protein domains requires the folding or structuring of those regions such that they can establish well-defined stoichiometric interactions. Since, in such a situation binding is coupled to folding, the energetics of those two events is reflected in the measured binding thermodynamics. In this protocol, we illustrate the thermodynamic differences between binding coupled to folding and binding independent of folding for the same protein. As an example, we use the HIV-1 envelope glycoprotein gp120 that contains structured as well as disordered domains. In the experiments presented, the binding of gp120 to molecules that bind to disordered regions and trigger structuring (CD4 or MAb 17b) and to molecules that bind to structured regions and do not induce conformational structuring (MAb b12) is discussed.
Asunto(s)
Calorimetría/métodos , Proteínas Intrínsecamente Desordenadas/química , Anticuerpos Monoclonales/metabolismo , Reacciones Antígeno-Anticuerpo , Antígenos CD4/metabolismo , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/inmunología , Humanos , Proteínas Intrínsecamente Desordenadas/metabolismo , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
In the version of this article originally published, data were incorrectly ascribed to monoclonal antibody CIS34 because of a labeling error. The data were generated with monoclonal antibody CIS04. Full details can be found in the correction notice.
RESUMEN
It has been shown that isothermal calorimetry is able to provide critical information regarding the kinetics of denaturation/aggregation of monoclonal antibodies at temperatures below Tm. Those measurements, however, required sophisticated specialized instrumentation. Here, we demonstrate that similar measurements can be performed using widely available conventional differential scanning calorimeters (DSC) when operated in isothermal scan mode. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal DSC at ten degrees below Tm. It is shown that a readily available instrument provides similar kinetic information and can become an important tool for determining the long term stability of biologics.
Asunto(s)
Anticuerpos Monoclonales/análisis , Rastreo Diferencial de Calorimetría/instrumentación , Rastreo Diferencial de Calorimetría/métodos , Anticuerpos Monoclonales/química , Calorimetría/métodos , Cinética , Desnaturalización Proteica , TemperaturaRESUMEN
The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.
Asunto(s)
Antígenos CD4/metabolismo , Proteína gp120 de Envoltorio del VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Antígenos CD4/genética , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , Infecciones por VIH/virología , Humanos , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Many one-dimensional (1D) nanostructures are constructed by self-assembly of peptides or peptide conjugates containing a short ß-sheet sequence as the core building motif essential for the intermolecular hydrogen bonding that promotes directional, anisotropic growth of the resultant assemblies. While this molecular engineering strategy has led to the successful production of a plethora of bioactive filamentous ß-sheet assemblies for interfacing with biomolecules and cells, concerns associated with effective presentation of α-helical epitopes and their function preservation have yet to be resolved. In this context, we report on the direct conjugation of the protein A mimicking peptide Z33, a motif containing two α-helices, to linear hydrocarbons to create self-assembling immuno-amphiphiles (IAs). Our results suggest that the resulting amphiphilic peptides can, despite lacking the essential ß-sheet segment, effectively associate under physiological conditions into supramolecular immunofibers (IFs) while preserving their native α-helical conformation. Isothermal titration calorimetry (ITC) measurements confirmed that these self-assembling immunofibers can bind to the human immunoglobulin G class 1 (IgG1) with high specificity at pH 7.4, but with significantly weakened binding at pH 2.8. We further demonstrated the accessibility of Z33 ligand in the immunofibers using transmission electron microscopy (TEM) and confocal imaging. We believe these results shed important light into the supramolecular engineering of α-helical peptides into filamentous assemblies that may possess an important potential for antibody isolation.
Asunto(s)
Biomimética/métodos , Inmunoglobulina G/metabolismo , Péptidos/química , Péptidos/inmunología , Secuencia de Aminoácidos , Calorimetría , Fluorescencia , Ligandos , Nanoestructuras/química , Nanoestructuras/ultraestructura , Unión Proteica , TermodinámicaRESUMEN
Different factors affect the long term stability of monoclonal antibodies, among them denaturation or partial denaturation that is often followed by aggregation. Isothermal calorimetry is capable of quantifying the kinetics of denaturation/aggregation of an antibody by measuring the heat that is released or absorbed by the process over a period of days or weeks, at temperatures below its denaturation temperature, Tm. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal calorimetry at different concentrations in four different formulation buffers. The measurements were performed at ten degrees below Tm, as determined by differential scanning calorimetry. The formation of aggregates was also followed by size exclusion chromatography at 5⯰C, 25⯰C and 40⯰C over a period of 8-36 weeks. It was observed that the rates measured by isothermal calorimetry correlate quantitatively with those measured by size exclusion chromatography. Since isothermal calorimetry experiments are performed over a period of ten days, it can become a valuable tool for a fast prediction of the best formulations.
Asunto(s)
Anticuerpos Anti-VIH/química , VIH-1/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Apraxia Ideomotora , Calorimetría/métodos , Rastreo Diferencial de Calorimetría/métodos , Calor , Humanos , Agregado de Proteínas , Desnaturalización Proteica , Estabilidad ProteicaRESUMEN
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design.
Asunto(s)
Vacunas contra la Malaria/inmunología , Malaria/inmunología , Parásitos/inmunología , Proteínas Protozoarias/química , Animales , Anticuerpos Monoclonales , Anticuerpos Antiprotozoarios/inmunología , Humanos , Ratones , Plasmodium falciparum/inmunologíaRESUMEN
The original version of this Article contained an error in the spelling of the author Amos B. Smith, III, which was incorrectly given as Amos B. SmithIII. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMEN
The entry of HIV-1 into target cells is mediated by the viral envelope glycoproteins (Env). Binding to the CD4 receptor triggers a cascade of conformational changes in distant domains that move Env from a functionally "closed" State 1 to more "open" conformations, but the molecular mechanisms underlying allosteric regulation of these transitions are still elusive. Here, we develop chemical probes that block CD4-induced conformational changes in Env and use them to identify a potential control switch for Env structural rearrangements. We identify the gp120 ß20-ß21 element as a major regulator of Env transitions. Several amino acid changes in the ß20-ß21 base lead to open Env conformations, recapitulating the structural changes induced by CD4 binding. These HIV-1 mutants require less CD4 to infect cells and are relatively resistant to State 1-preferring broadly neutralizing antibodies. These data provide insights into the molecular mechanism and vulnerability of HIV-1 entry.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Antígenos CD4/metabolismo , Células HEK293 , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/fisiología , Humanos , Sondas Moleculares , Unión Proteica , Conformación Proteica , Conformación Proteica en Lámina betaRESUMEN
Lipoate is an essential cofactor for enzymes that are important for central metabolism and other processes. In malaria parasites, scavenged lipoate from the human host is required for survival. The Plasmodium falciparum mitochondrion contains two enzymes (PfLipL1 and PfLipL2) that are responsible for activating mitochondrial proteins through the covalent attachment of lipoate (lipoylation). Lipoylation occurs via a novel redox-gated mechanism that remains poorly understood. We show that PfLipL1 functions as a redox switch that determines which downstream proteins will be activated. Based on the lipoate redox state, PfLipL1 either functions as a canonical lipoate ligase or as a lipoate activating enzyme which works in conjunction with PfLipL2. We demonstrate that PfLipL2 is a lipoyltransferase and is a member of a novel clade of lipoate attachment enzymes. We show that a LipL2 enzyme from Chlamydia trachomatis has similar activity, demonstrating conservation between intracellular pathogens from different phylogenetic kingdoms and supporting the hypothesis that an early ancestor of malaria parasites once contained a chlamydial endosymbiont. Redox-dependent lipoylation may regulate processes such as central metabolism and oxidative defense pathways.
Asunto(s)
Lipoilación/genética , Lipoilación/fisiología , Chlamydia/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Nucleotidiltransferasas , Oxidación-Reducción , Péptido Sintasas/genética , Plasmodium/metabolismo , Plasmodium falciparum/genética , Proteínas Protozoarias/metabolismo , Alineación de SecuenciaRESUMEN
The HIV-1 envelope (Env) spike is a conformational machine that transitions between prefusion (closed, CD4- and CCR5-bound) and postfusion states to facilitate HIV-1 entry into cells. Although the prefusion closed conformation is a potential target for inhibition, development of small-molecule leads has been stymied by difficulties in obtaining structural information. Here, we report crystal structures at 3.8-Å resolution of an HIV-1-Env trimer with BMS-378806 and a derivative BMS-626529 for which a prodrug version is currently in Phase III clinical trials. Both lead candidates recognized an induced binding pocket that was mostly excluded from solvent and comprised of Env elements from a conserved helix and the ß20-21 hairpin. In both structures, the ß20-21 region assumed a conformation distinct from prefusion-closed and CD4-bound states. Together with biophysical and antigenicity characterizations, the structures illuminate the allosteric and competitive mechanisms by which these small-molecule leads inhibit CD4-induced structural changes in Env.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , Piperazinas/química , Bibliotecas de Moléculas Pequeñas/química , Triazoles/química , Internalización del Virus/efectos de los fármacos , Cristalografía por Rayos X , Proteína gp120 de Envoltorio del VIH/antagonistas & inhibidores , Proteína gp41 de Envoltorio del VIH/antagonistas & inhibidores , Modelos Moleculares , Piperazinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Triazoles/farmacologíaRESUMEN
The structural stability of proteins has been traditionally studied under conditions in which the folding/unfolding reaction is reversible, since thermodynamic parameters can only be determined under these conditions. Achieving reversibility conditions in temperature stability experiments has often required performing the experiments at acidic pH or other nonphysiological solvent conditions. With the rapid development of protein drugs, the fastest growing segment in the pharmaceutical industry, the need to evaluate protein stability under formulation conditions has acquired renewed urgency. Under formulation conditions and the required high protein concentration (â¼100 mg/mL), protein denaturation is irreversible and frequently coupled to aggregation and precipitation. In this article, we examine the thermal denaturation of hen egg white lysozyme (HEWL) under irreversible conditions and concentrations up to 100 mg/mL using several techniques, especially isothermal calorimetry which has been used to measure the enthalpy and kinetics of the unfolding and aggregation/precipitation at 12°C below the transition temperature measured by DSC. At those temperatures the rate of irreversible protein denaturation and aggregation of HEWL is measured to be on the order of 1 day-1 . Isothermal calorimetry appears a suitable technique to identify buffer formulation conditions that maximize the long term stability of protein drugs.
Asunto(s)
Calorimetría/métodos , Desnaturalización Proteica , Estabilidad Proteica , Animales , Pollos , Muramidasa/análisis , Muramidasa/química , Muramidasa/metabolismo , Muramidasa/efectos de la radiación , Agregado de Proteínas/fisiología , TermodinámicaRESUMEN
The enthalpic and entropic contributions to the binding affinity of drug candidates have been acknowledged to be important determinants of the quality of a drug molecule. These quantities, usually summarized in the thermodynamic signature, provide a rapid assessment of the forces that drive the binding of a ligand. Having access to the thermodynamic signature in the early stages of the drug discovery process will provide critical information towards the selection of the best drug candidates for development. In this paper, the Enthalpy Screen technique is presented. The enthalpy screen allows fast and accurate determination of the binding enthalpy for hundreds of ligands. As such, it appears to be ideally suited to aid in the ranking of the hundreds of hits that are usually identified after standard high throughput screening.
Asunto(s)
Inhibidores de la Proteasa del VIH/química , Proteasa del VIH/química , VIH-1/enzimología , Termodinámica , Evaluación Preclínica de Medicamentos/métodosRESUMEN
The optimization, based on computational, thermodynamic, and crystallographic data, of a series of small-molecule ligands of the Phe43 cavity of the envelope glycoprotein gp120 of human immunodeficiency virus (HIV) has been achieved. Importantly, biological evaluation revealed that the small-molecule CD4 mimics (4-7) inhibit HIV-1 entry into target cells with both significantly higher potency and neutralization breadth than previous congeners, while maintaining high selectivity for the target virus. Their binding mode was characterized via thermodynamic and crystallographic studies.
RESUMEN
Biologics exist in equilibrium between native, partially denatured, and denatured conformational states. The population of any of these states is dictated by their Gibbs energy and can be altered by changes in physical and solution conditions. Some conformations have a tendency to self-associate and aggregate, an undesirable phenomenon in protein therapeutics. Conformational equilibrium and self-association are linked thermodynamic functions. Given that any associative reaction is concentration dependent, conformational stability studies performed at different protein concentrations can provide early clues to future aggregation problems. This analysis can be applied to the selection of protein variants or the identification of better formulation solutions. In this review, we discuss three different aggregation situations and their manifestation in the observed conformational equilibrium of a protein.
Asunto(s)
Productos Biológicos/química , Conformación Proteica , Desnaturalización Proteica , Proteínas/química , TermodinámicaRESUMEN
BACKGROUND: Differential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25°C, may become unreliable due to difficulties in the determination of ΔCp. METHODS: The combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions. RESULTS AND CONCLUSIONS: This paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔCp value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin.
Asunto(s)
Proteínas Bacterianas/química , Proteína gp120 de Envoltorio del VIH/química , Muramidasa/química , Termolisina/química , Animales , Bacillus/química , Rastreo Diferencial de Calorimetría , Pollos , VIH-1/química , Concentración de Iones de Hidrógeno , Cinética , Desnaturalización Proteica , Pliegue de Proteína , Temperatura , TermodinámicaRESUMEN
Small-molecule mimetics of the ß-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 µM.