RESUMEN
Aggregation of proteins is a critical quality attribute and a major concern during the purification of therapeutic proteins, like monoclonal antibodies. In-solution experiments applying different stress scenarios, e.g., mechanical, or physical stresses, can determine the overall conformational stability of the protein to enhance drug product shelf-life. Several groups have reported surface-induced unfolding and aggregation of monoclonal antibodies and their derivatives during cation exchange chromatography, which results in a two-peak elution behavior of the protein and its species. We have investigated universal influencing factors, like temperature and hold time, on this phenomenon. The formation of the second peak is a kinetic process, which is strongly influenced by temperature during the hold time. However, our main focus was the application of excipients and their influence on the two-peak elution behavior. We compared the on-column screening results with results obtained through a "traditional" in-solution screening using nanoDSF. Mostly, stabilizing excipients, like Sucrose, show their stabilizing abilities in both systems, but some discrepancies, e.g., using Arginine, between the two orthogonal techniques show the potential of the on-column screening system to lead to unexpected results, which would not necessarily be visible in in-solution experiments.
Asunto(s)
Anticuerpos Monoclonales , Excipientes , Cromatografía por Intercambio Iónico/métodos , Excipientes/química , Anticuerpos Monoclonales/química , Temperatura , CationesRESUMEN
The application of surfactants in liquid protein formulation is a common practice to protect proteins from liquid-air interface-induced protein aggregation. Typically, Polysorbate 20 or 80 are used, but degradation of these surfactants can result in particle formation and/or protein degradation. The purpose of the current study was to directly compare three alternative protein stabilizing molecules - Poloxamer 188, hydroxypropyl-cyclodextrin and a trehalose-based surfactant - to Polysorbate 80 for their capacities to reduce agitation-induced protein aggregation and particle formation; and furthermore, investigate their underlying protein stabilizing mechanisms. To this end, a small-volume, rapid agitation stress approach was used to quantify the molecules' abilities to stabilize two model proteins. This assay was presented to be a powerful tool to screen the protein stabilizing capability of surfactants using minimum of material and time. SEC, turbidity measurements and particle analysis showed an efficient protein stabilization of all tested surfactants as well as cyclodextrin. STD-NMR and dynamic surface tension measurements indicated the competitive surface adsorption to be the main protein stabilizing mechanism of the three surfactants tested. It might also play a role to some extent in the protein stabilization by HPßCD. However, additional mechanisms might also contribute to protein stabilization leaving room for further investigations.
Asunto(s)
Agregado de Proteínas , Tensoactivos , Tensoactivos/química , Polisorbatos/química , Excipientes/química , 2-Hidroxipropil-beta-Ciclodextrina , Proteínas/químicaRESUMEN
It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.
Asunto(s)
Archaea/química , Archaea/fisiología , Extremófilos/química , Extremófilos/fisiología , Lípidos de la Membrana/química , Aclimatación/fisiología , Ambientes Extremos , Calor , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Lípidos de la Membrana/síntesis química , Modelos Moleculares , Estructura Molecular , Difracción de Neutrones , Permeabilidad , Presión , Dispersión del Ángulo Pequeño , Espectrometría de Fluorescencia , Escualeno/análogos & derivados , Escualeno/química , Terpenos/química , Difracción de Rayos XRESUMEN
Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Organofosfatos/farmacología , Proteínas del Envoltorio Viral/efectos de los fármacos , Fosfatasa Ácida/química , Fosfatasa Ácida/metabolismo , Amiloide/antagonistas & inhibidores , Fármacos Anti-VIH/química , Fármacos Anti-VIH/farmacología , Arginina/química , Betacoronavirus/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/química , Membrana Celular/química , Membrana Celular/efectos de los fármacos , Membrana Celular/virología , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Humanos , Lípidos/química , Lisina/química , Espectroscopía de Resonancia Magnética , Organofosfatos/química , SARS-CoV-2 , Proteínas de Secreción de la Vesícula Seminal/química , Proteínas de Secreción de la Vesícula Seminal/metabolismo , Relación Estructura-Actividad , Proteínas del Envoltorio Viral/metabolismo , Virus Zika/efectos de los fármacosRESUMEN
Autophagy mediates the degradation of damaged proteins, organelles and pathogens, and plays a key role in health and disease. Thus, the identification of new mechanisms involved in the regulation of autophagy is of major interest. In particular, little is known about the role of lipids and lipid-binding proteins in the early steps of autophagosome biogenesis. Using target-agnostic, high-content, image-based identification of indicative phenotypic changes induced by small molecules, we have identified autogramins as a new class of autophagy inhibitor. Autogramins selectively target the recently discovered cholesterol transfer protein GRAM domain-containing protein 1A (GRAMD1A, which had not previously been implicated in autophagy), and directly compete with cholesterol binding to the GRAMD1A StART domain. GRAMD1A accumulates at sites of autophagosome initiation, affects cholesterol distribution in response to starvation and is required for autophagosome biogenesis. These findings identify a new biological function of GRAMD1A and a new role for cholesterol in autophagy.
Asunto(s)
Autofagosomas/metabolismo , Proteínas de la Membrana/metabolismo , Autofagosomas/efectos de los fármacos , Autofagia/efectos de los fármacos , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales CultivadasRESUMEN
Oncogenic Ras mutations occur in more than 30 % of human cancers. K-Ras4B is the most frequently mutated isoform of Ras proteins. Development of effective K-Ras4B inhibitors has been challenging, hence new approaches to inhibit this oncogenic protein are urgently required. The polybasic domain of K-Ras4B with its stretch of lysine residues is essential for its plasma membrane targeting and localization. Employing CD and fluorescence spectroscopy, confocal fluorescence, and atomic force microscopy we show that the molecular tweezer CLR01 is able to efficiently bind to the lysine stretch in the polybasic domain of K-Ras4B, resulting in dissociation of the K-Ras4B protein from the lipid membrane and disintegration of K-Ras4B nanoclusters in the lipid bilayer. These results suggest that targeting of the polybasic domain of K-Ras4B by properly designed tweezers might represent an effective strategy for inactivation of K-Ras4B signaling.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Membrana Celular/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Organofosfatos/química , Proteínas Proto-Oncogénicas p21(ras)/química , Simulación por Computador , Humanos , Mutación , Nanoestructuras/química , Conformación Proteica , TermodinámicaRESUMEN
Signaling of N-Ras and K-Ras4B proteins depends strongly on their correct localization in the cell membrane. In vivo studies suggest that intermolecular interactions foster the self-association of both N-Ras and K-Ras4B and the formation of nanoclusters in the cell membrane. As sites for effector binding, nanocluster formation is thought to be essential for effective signal transmission of both N-Ras and K-Ras4B. To shed more light on the spatial arrangement and mechanism underlying the proposed cross-talk between spatially segregated Ras proteins, the simultaneous localization of N-Ras and K-Ras4B and their effect on the lateral organization of a heterogeneous model biomembrane has been studied by using AFM and FRET methodology. It is shown that, owing to the different natures of their membrane anchor systems, N-Ras and K-Ras4B not only avoid assembly in bulk solution and do not colocalize, but rather form individual nanoclusters that diffuse independently in the fluid membrane plane.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Lipoproteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Liposomas Unilamelares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Colesterol/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Membrana Dobles de Lípidos/química , Microdominios de Membrana , Fosfatidilcolinas/química , Liposomas Unilamelares/químicaRESUMEN
Plasma membrane localization of myristoylated c-Src, a proto-oncogene protein-tyrosine kinase, is required for its signaling activity. Recent studies proposed that UNC119 protein functions as a solubilizing factor for myristoylated proteins, thereby regulating their subcellular distribution and signaling. The underlying molecular mechanism by which UNC119 regulates the membrane binding of c-Src has remained elusive. By combining different biophysical techniques, we have found that binding of a myristoylated c-Src-derived N-terminal peptide (Myr-Src) by UNC119A results in a reduced membrane binding affinity of the peptide, due to the competition of binding to membranes. The dissociation of Myr-Src from membranes is facilitated in the presence of UNC119A, as a consequence of which the clustering propensity of this peptide on the membrane is partially impaired. By these means, UNC119A is able to regulate c-Src spatially in the cytoplasm and on cellular membranes, and this has important implications for its cellular signaling.
RESUMEN
For over 50 years, it has been known that the mitosis of eukaryotic cells is inhibited already at high hydrostatic pressure conditions of 30 MPa. This effect has been attributed to the disorganization of microtubules, the main component of the spindle apparatus. However, the structural details of the depolymerization and the origin of the pressure sensitivity have remained elusive. It has also been a puzzle how complex organisms could still successfully inhabit extreme high-pressure environments such as those encountered in the depth of oceans. We studied the pressure stability of microtubules at different structural levels and for distinct dynamic states using high-pressure Fourier-transform infrared spectroscopy and Synchrotron small-angle x-ray scattering. We show that microtubules are hardly stable under abyssal conditions, where pressures up to 100 MPa are reached. This high-pressure sensitivity can be mainly attributed to the internal voids and packing defects in the microtubules. In particular, we show that lateral and longitudinal contacts feature different pressure stabilities, and they define also the pressure stability of tubulin bundles. The intactness of both contact types is necessary for the functionality of microtubules in vivo. Despite being known to dynamically stabilize microtubules and prevent their depolymerization, we found that the anti-cancer drug taxol and the accessory protein MAP2c decrease the pressure stability of microtubule protofilaments. Moreover, we demonstrate that the cellular environment itself is a crowded place and accessory proteins can increase the pressure stability of microtubules and accelerate their otherwise highly pressure-sensitive de novo formation.
Asunto(s)
Microtúbulos/metabolismo , Presión , Animales , Encéfalo/citología , Bovinos , Cinética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , RatasRESUMEN
Ras proteins are oncoproteins which play a pivotal role in cellular signaling pathways. All Ras proteins' signaling strongly depends on their correct localization in the cell membrane. Over 30% of cancers are driven by mutant Ras proteins, and KRas4B is the Ras isoform most frequently mutated. C6-ceramide has been shown to inhibit the growth activity of KRas4B mutated cells. However, the mechanism underlying this inhibition remains elusive. Here, we established a heterogeneous model biomembrane containing C6-ceramide. C6-ceramide incorporation does not disrupt the lipid membrane. Addition of KRas4B leads to drastic changes in the lateral membrane organization of the membrane, however. In contrast to the partitioning behavior in other membranes, KRas4B forms small, monodisperse nanoclusters dispersed in a fluid-like environment, in all likelihood induced by some kind of lipid sorting mechanism. Fluorescence cross-correlation data indicate no direct interaction between C6-ceramide and KRas4B, suggesting that KRas4B essentially recruits other lipids. A FRET-based binding assay reveals that the stability of KRas4B proteins inserted into the membrane containing C6-ceramide is reduced. Based on the combined results obtained, we postulate a molecular mechanism for the inhibition of KRas4B mutated cells' activity through C6-ceramide.
Asunto(s)
Ceramidas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Ceramidas/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Microscopía de Fuerza Atómica , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas p21(ras)/química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismoRESUMEN
The structural dynamics of a DNA hairpin (Hp) are studied in the absence and presence of the two natural osmolytes trimethylamine-N-oxide (TMAO) and urea at ambient and extreme environmental conditions, including high pressures and high temperatures, by using single-molecule Förster resonance energy transfer and fluorescence correlation spectroscopy. The effect of pressure on the conformational dynamics of the DNA Hp is investigated on a single-molecule level, providing novel mechanistic insights into its conformational conversions. Different from canonical DNA duplex structures of similar melting points, the DNA Hp is found to be rather pressure sensitive. The combined temperature and pressure dependent data allow dissection of the folding free energy into its enthalpic, entropic, and volumetric contributions. The folded conformation is effectively stabilized by the compatible osmolyte TMAO not only at high temperatures, but also at high pressures and in the presence of the destabilizing co-solute urea.
Asunto(s)
ADN/química , Metilaminas/química , Urea/química , Transferencia Resonante de Energía de Fluorescencia , Calor , Conformación de Ácido Nucleico , Concentración Osmolar , Presión , TermodinámicaRESUMEN
The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.
Asunto(s)
Membrana Celular/metabolismo , Metabolismo de los Lípidos , Membranas Artificiales , Proteínas ras/química , Proteínas ras/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Humanos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
The ubiquitous Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. The conformational space of this protein needs to be explored to shed more light on the mechanism of target recognition and protein function. Here, we used pressure modulation in combination with FTIR spectroscopy to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to its ground state, which are supposed to be functionally relevant in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the hypervariable region (HVR) of the signaling peptide K-Ras4B as a binding partner. We demonstrate that the conformational dynamics of CaM is vastly affected by binding of both Ca2+ ions and the lipidated signaling peptide K-Ras4B. Moreover, we could uncover conformational substates of CaM by pressure perturbation that are partially unfolded and more solvated and conceivably facilitate target recognition by exposing the required binding surfaces.
RESUMEN
We studied the combined effects of pressure (0.1-200â MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high-pressure stopped-flow system with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about -14â cm(3) mol(-1) ). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45-60â kJ mol(-1) ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.
Asunto(s)
Amiloide/metabolismo , Esterasas/metabolismo , Amiloide/química , Biocatálisis , Hidrólisis , Presión Hidrostática , Cinética , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Teoría Cuántica , Espectroscopía Infrarroja por Transformada de Fourier , TemperaturaRESUMEN
In a combined chemical-biological and biophysical approach we explored the membrane partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into membrane systems of different complexity, ranging from one-component lipid bilayers and anionic binary and ternary heterogeneous membrane systems even up to partitioning studies on protein-free and protein-containing giant plasma membrane vesicles (GPMVs). To yield a pictorial view of the localization process, imaging using confocal laser scanning and atomic force microscopy was performed. The results reveal pronounced isoform-specific differences regarding the lateral distribution and formation of protein-rich membrane domains. Line tension is one of the key parameters controlling not only the size and dynamic properties of segregated lipid domains but also the partitioning process of N-Ras that acts as a lineactant. The formation of N-Ras protein clusters is even recorded for almost vanishing hydrophobic mismatch. Conversely, for K-Ras4B, selective localization and clustering are electrostatically mediated by its polybasic farnesylated C-terminus. The formation of K-Ras4B clusters is also observed for the multi-component GPMV membrane, i.e., it seems to be a general phenomenon, largely independent of the details of the membrane composition, including the anionic charge density of lipid headgroups. Our data indicate that unspecific and entropy-driven membrane-mediated interactions play a major role in the partitioning behavior, thus relaxing the need for a multitude of fine-tuned interactions. Such a scenario seems also to be reasonable recalling the high dynamic nature of cellular membranes. Finally, we note that even relatively simple models of heterogeneous membranes are able to reproduce many of the properties of much more complex biological membranes.
Asunto(s)
Lípidos/química , Lipoproteínas/química , Adsorción , Microscopía de Fuerza Atómica , Microscopía FluorescenteRESUMEN
Ras proteins are small GTPases and are involved in transmitting signals that control cell growth, differentiation, and proliferation. Since the cell cytoplasm is crowded with different macromolecules, understanding the translational dynamics of Ras proteins in crowded environments is crucial to yielding deeper insight into their reactivity and function. Herein, the translational dynamics of lipidated N-Ras and K-Ras4B is studied in the bulk and in the presence of a macromolecular crowder (Ficoll) and the compatible osmolyte and microcrowder sucrose by fluorescence correlation spectroscopy. The results reveal that N-Ras forms dimers due to the presence of its lipid moiety in the hypervariable region, whereas K-Ras4B remains in its monomeric form in the bulk. Addition of a macromolecular crowding agent gradually favors clustering of the Ras proteins. In 20â wt % Ficoll N-Ras forms trimers and K-Ras4B dimers. Concentrations of sucrose up to 10â wt % foster formation of N-Ras trimers and K-Ras dimers as well. The results can be rationalized in terms of the excluded-volume effect, which enhances the association of the proteins, and, for the higher concentrations, by limited-hydration conditions. The results of this study shed new light on the association state of these proteins in a crowded environment. This is of particular interest for the Ras proteins, because their solution state-monomeric or clustered-influences their membrane-partitioning behavior and their interplay with cytosolic interaction partners.
Asunto(s)
Proteínas ras/química , Difusión , Ficoll/química , Hidrodinámica , Microscopía Confocal , Prenilación de Proteína , Espectrometría de Fluorescencia , Sacarosa/química , Proteínas ras/metabolismoRESUMEN
Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.
Asunto(s)
Amiloide/antagonistas & inhibidores , Fármacos Anti-VIH/farmacología , Antimetabolitos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Organofosfatos/farmacología , Semen/efectos de los fármacos , Transmisión de Enfermedad Infecciosa/prevención & control , Infecciones por VIH/prevención & control , Infecciones por VIH/transmisión , Humanos , Masculino , Semen/química , Semen/virologíaRESUMEN
Not only drastic temperature- but also pressure-induced perturbations of membrane organization pose a serious challenge to the biological cell. Although high hydrostatic pressure significantly influences the structural properties and thus functional characteristics of cells, this has not prevented life from invading the high pressure habitats of marine depths where pressures up to the 100 MPa level are encountered. Here, the temperature- and pressure-dependent structure and phase behavior of giant plasma membrane vesicles have been explored in the absence and presence of membrane proteins using a combined spectroscopic and microscopic approach. Demixing into extended liquid-ordered and liquid-disordered domains is observed over a wide range of temperatures and pressures. Only at pressures beyond 200 MPa a physiologically unfavorable all gel-like ordered lipid phase is reached at ambient temperature. This is in fact the pressure range where the membrane-protein function has generally been observed to cease, thereby shedding new light on the possible origin of this observation.
Asunto(s)
Membrana Celular/química , Presión , Temperatura , Animales , Línea Celular Tumoral , Proteínas de la Membrana/química , RatasRESUMEN
Pressure perturbation calorimetry (PPC) is an efficient technique to study the volumetric properties of biomolecules in solution. In PPC, the coefficient of thermal expansion of the partial volume of the biomolecule is deduced from the heat consumed or produced after small isothermal pressure-jumps. The expansion coefficient strongly depends on the interaction of the biomolecule with the solvent or cosolvent as well as on its packing and internal dynamic properties. This technique, complemented with molecular acoustics and densimetry, provides valuable insights into the basic thermodynamic properties of solvation and volume effects accompanying interactions, reactions and phase transitions of biomolecular systems. After outlining the principles of the technique, we present representative examples on protein folding, including effects of cosolvents and crowding, together with a discussion of the interpretation, and further applications.
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Calorimetría/métodos , Pliegue de Proteína , Proteínas/química , Muramidasa/química , Poliestirenos/química , Ribonucleasa Pancreática/química , Solventes/química , Temperatura , Termodinámica , Agua/químicaRESUMEN
Owing to the presence of various types of osmolytes in the cellular environment, this study focuses on the impact of stabilizing (TMAO and betaine) as well as destabilizing (urea) cosolvents on the aggregation and fibrillation reaction of the highly amyloidogenic islet amyloid polypeptide (IAPP). IAPP is associated with type-2 diabetes mellitus and is responsible for the disease accompanying ß-cell membrane permeabilization and final ß-cell loss. To reveal the impact of the cosolvents on the aggregation kinetics, conformational and morphological changes upon IAPP fibrillation, Thioflavin T fluorescence spectroscopy, atomic force microscopy and attenuated total reflection Fourier-transform infrared spectroscopy were applied. For TMAO, and less pronounced for betaine, a decrease of the growth rate of fibrils is observed, whereas the lag phase remains essentially unchanged, indicating the ability of the compatible solutes to stabilize large oligomeric and protofibrillar structures and therefore hamper fibril elongation. Conversely, urea displays concentration-dependent prolongation of the lag phase, indicating stabilization of IAPP in its unfolded monomeric state, hence leading to retardation of IAPP nuclei formation. Mixtures of urea with TMAO, and to a lesser extent with betaine, exhibit a counteractive effect. TMAO is able to fully compensate the prolonged lag phase induced by urea. This strongly matches the findings of a counteraction of TMAO and urea in protein folding and unfolding experiments. The data also reveal that the influence of these cosolvents is only on the aggregation kinetics without markedly changing the final IAPP fibrillar morphology, i.e., the solution structure and cosolvent composition essentially affect the kinetics of the fibrillation process only.