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1.
Molecules ; 28(6)2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36985597

RESUMO

In the context of targeted radionuclide therapy, antibody-chelator conjugates (ACCs) are an evolving class of antibody-related drugs with promising applications as tumor-targeted pharmaceuticals. Generally, a typical ACC consists of a recombinant monoclonal antibody (mAb) coupled to radionuclide via a chelating agent. Characterizing the ACC structure represents an analytical challenge since various impurities must be constantly monitored in the presence of formulation components during the quality control (QC) process. In this contribution, a reliable method devoted to the monitoring of an ACC sample, and its small molecule-related synthesis impurities, has been developed via liquid chromatography (LC). A problem-solving approach of common analytical issues was used to highlight some major issues encountered during method development. This included separation of poorly retained impurities (issue #1); interferences from the formulation components (issue #2); analysis of impurities in presence of ACC at high concentration (issue #3); and recovery of impurities during the whole analytical procedure (issue #4). To the best of our knowledge, this is the first time that a chromatographic method for the analysis of ACC synthesis impurities is presented. In addition, the developed approach has the potential to be more widely applied to the characterization of similar ACCs and other antibody-related drugs.


Assuntos
Imunoconjugados , Cromatografia Líquida , Imunoconjugados/química , Anticorpos Monoclonais/química , Radioisótopos , Cromatografia Líquida de Alta Pressão/métodos
2.
Phys Chem Chem Phys ; 22(6): 3734-3743, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32010904

RESUMO

The chaperonin system GroEL-GroES is present in all kingdoms of life and rescues proteins from improper folding and aggregation upon internal and external stress conditions, including high temperatures and pressures. Here, we set out to explore the thermo- and piezostability of GroEL, GroES and the GroEL-GroES complex in the presence of cosolvents, nucleotides and salts employing quantitative FTIR spectroscopy and small-angle X-ray scattering. Owing to its high biological relevance and lack of data, our focus was especially on the effect of pressure on the chaperonin system. The experimental results reveal that the GroEL-GroES complex is remarkably temperature stable with an unfolding temperature beyond 70 °C, which can still be slightly increased by compatible cosolutes like TMAO. Conversely, the pressure stability of GroEL and hence the GroEL-GroES complex is rather limited and much less than that of monomeric proteins. Whereas GroES is pressure stable up to ∼5 kbar, GroEl and the GroEl-GroES complex undergo minor structural changes already beyond 1 kbar, which can be attributed to a dissociation-induced conformational drift. Quite unexpectedly, no significant unfolding of GroEL is observed even up to 10 kbar, however, i.e., the subunits themselves are very pressure stable. As for the physiological relevance, the structural integrity of the chaperonin system is retained in a relatively narrow pressure range, from about 1 to 1000 bar, which is just the pressure range encountered by life on Earth.


Assuntos
Chaperonina 10/química , Chaperonina 60/química , Meio Ambiente , Pressão , Estabilidade Proteica , Temperatura
3.
Chem Soc Rev ; 48(14): 3946-3996, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31192324

RESUMO

One of the grand challenges of biophysical chemistry is to understand the principles that govern protein misfolding and aggregation, which is a highly complex process that is sensitive to initial conditions, operates on a huge range of length- and timescales, and has products that range from protein dimers to macroscopic amyloid fibrils. Aberrant aggregation is associated with more than 25 diseases, which include Alzheimer's, Parkinson's, Huntington's, and type II diabetes. Amyloid aggregation has been extensively studied in the test tube, therefore under conditions that are far from physiological relevance. Hence, there is dire need to extend these investigations to in vivo conditions where amyloid formation is affected by a myriad of biochemical interactions. As a hallmark of neurodegenerative diseases, these interactions need to be understood in detail to develop novel therapeutic interventions, as millions of people globally suffer from neurodegenerative disorders and type II diabetes. The aim of this review is to document the progress in the research on amyloid formation from a physicochemical perspective with a special focus on the physiological factors influencing the aggregation of the amyloid-ß peptide, the islet amyloid polypeptide, α-synuclein, and the hungingtin protein.


Assuntos
Amiloide/química , Agregados Proteicos , Agregação Patológica de Proteínas , Animais , Humanos
4.
Biophys J ; 114(5): 1080-1090, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29539395

RESUMO

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.


Assuntos
Microtúbulos/metabolismo , Pressão , Animais , Encéfalo/citologia , Bovinos , Cinética , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Ratos
5.
Phys Rev Lett ; 121(3): 038101, 2018 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-30085800

RESUMO

The influence of natural cosolvent mixtures on the pressure-dependent structure and protein-protein interaction potential of dense protein solutions is studied and analyzed using small-angle X-ray scattering in combination with a liquid-state theoretical approach. The deep-sea osmolyte trimethylamine-N-oxide is shown to play a crucial and singular role in its ability to not only guarantee sustainability of the native protein's folded state under harsh environmental conditions, but it also controls water-mediated intermolecular interactions at high pressure, thereby preventing contact formation and hence aggregation of proteins.


Assuntos
Modelos Químicos , Muramidase/química , Água/química , Pressão Hidrostática , Metilaminas/química , Concentração Osmolar , Espalhamento a Baixo Ângulo , Soluções , Difração de Raios X
6.
Chemphyschem ; 18(2): 189-197, 2017 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-27813294

RESUMO

Tubulin is one of the main components of the cytoskeleton and can be found in nearly all eukaryotic cells. In this study, we explored the effects of kosmotropic and chaotropic osmolytes, such as trimethylamine-N-oxide (TMAO) and the metabolic waste product urea, as well as the crowding agents Ficoll and sucrose on the polymerization reaction of α/ß-tubulin. Time-dependent turbidimetry and fluorescence anisotropy experiments were performed to explore the kinetics of the polymerization reaction. Under different solvent conditions, diverse changes in the lag time, the half-life of the polymerization reaction, and the critical concentration of the polymerization reaction were observed. The apparent growth rate of the formation of microtubules was dramatically decreased in the presence of urea but significantly increased in the presence of TMAO. Measurements using mixtures of these two cosolvents showed that TMAO was able to counteract the deteriorating effect of urea on the polymerization reaction of tubulin. To create a more cell-like environment, Ficoll was added as a macromolecular crowding agent. The presence of 10 wt % Ficoll increased the apparent growth rate by one order of magnitude. Our results clearly show that the polymerization of tubulin is very sensitive to the surrounding solvent.


Assuntos
Metilaminas/química , Tubulina (Proteína)/química , Ureia/química , Ficoll/química , Cinética , Substâncias Macromoleculares/química , Osmose , Polimerização , Solventes/química , Sacarose/química
7.
Chemphyschem ; 18(21): 2951-2972, 2017 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-28810057

RESUMO

The free energy and conformational landscape of biomolecular systems as well as biochemical reactions depend not only on temperature and pressure, but also on the particular solution conditions. Such conditions include the effects of cosolvents (for example osmolytes) and macromolecular crowding, which are crucial components to understand the energetics and kinetics of biological processes in living system. Such conditions are also important for the understanding of many debilitating diseases, such as those where misfolding and amyloid formation of proteins are involved. Moreover, understanding their effects on biomolecular processes is prerequisite for designing industrially relevant enzymatic reactions, which seldom take place under neat conditions. Here, we review and discuss experimental and theoretical studies on the characterization of cosolvent and crowding induced effects in biologically relevant systems, approaching even the complexity of living organisms. In particular, we focus on cosolvent and crowding effects on the conformational equilibrium and folding kinetics of proteins and nucleic acids as well as on enzymatic reactions, including their effects on the temperature and pressure dependence of these processes. By presenting a few representative examples, we show how such effects are unveiled and described in thermodynamic and kinetic terms.


Assuntos
Ácidos Nucleicos/química , Proteínas/química , Animais , Humanos , Cinética , Substâncias Macromoleculares/química , Pressão , Solventes/química , Termodinâmica
8.
Phys Chem Chem Phys ; 19(17): 10738-10747, 2017 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-28094373

RESUMO

Huntington's disease is caused by a CAG trinucleotide expansion mutation in the Huntingtin gene that leads to an artificially long polyglutamine sequence in the Huntingtin protein. A key feature of the disease is the intracellular aggregation of the Huntingtin exon 1 protein (Httex1) into micrometer sized inclusion bodies. The aggregation process of Httex1 has been extensively studied in vitro, however, the crucial early events of nucleation and aggregation in the cell remain elusive. Here, we studied the conformational dynamics and self-association of Httex1 by in-cell experiments using laser-induced temperature jumps and analytical ultracentrifugation. Both short and long polyglutamine variants of Httex1 underwent an apparent temperature-induced conformational collapse. The temperature jumps generated a population of kinetically trapped species selectively for the longer polyglutamine variants of Httex1 proteins. Their occurrence correlated with the formation of inclusion bodies suggesting that such species trigger further self-association.


Assuntos
Proteína Huntingtina/química , Proteína Huntingtina/metabolismo , Agregação Patológica de Proteínas/fisiopatologia , Proteína Huntingtina/genética , Doença de Huntington/fisiopatologia , Técnicas In Vitro , Corpos de Inclusão/metabolismo , Lasers , Modelos Moleculares , Mutação , Peptídeos/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Temperatura , Ultracentrifugação
9.
Angew Chem Int Ed Engl ; 56(9): 2302-2306, 2017 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-28102930

RESUMO

Folding of ribonucleic acids (RNAs) is driven by several factors, such as base pairing and stacking, chain entropy, and ion-mediated electrostatics, which have been studied in great detail. However, the power of background molecules in the cellular milieu is often neglected. Herein, we study the effect of common osmolytes on the folding equilibrium of a hairpin-structured RNA and, using pressure perturbation, provide novel thermodynamic and volumetric insights into the modulation mechanism. The presence of TMAO causes an increased thermal stability and a more positive volume change for the helix-to-coil transition, whereas urea destabilizes the hairpin and leads to an increased expansibility of the unfolded state. Further, we find a strong interplay between water, salt, and osmolyte in driving the thermodynamics and defining the temperature and pressure stability limit of the RNA. Our results support a universal working mechanism of TMAO and urea to (de)stabilize proteins and the RNA.


Assuntos
Metilaminas/química , Dobramento de RNA , Estabilidade de RNA , RNA/química , Ureia/química , Entropia , Conformação de Ácido Nucleico , Concentração Osmolar , Sais/química , Eletricidade Estática , Temperatura , Termodinâmica , Água/química
10.
Angew Chem Int Ed Engl ; 55(9): 3224-8, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26833452

RESUMO

Precise secondary and tertiary structure formation is critically important for the cellular functionality of ribonucleic acids (RNAs). RNA folding studies were mainly conducted in vitro, without the possibility of validating these experiments inside cells. Here, we directly resolve the folding stability of a hairpin-structured RNA inside live mammalian cells. We find that the stability inside the cell is comparable to that in dilute physiological buffer. On the contrary, the addition of in vitro artificial crowding agents, with the exception of high-molecular-weight PEG, leads to a destabilization of the hairpin structure through surface interactions and reduction in water activity. We further show that RNA stability is highly variable within cell populations as well as within subcellular regions of the cytosol and nucleus. We conclude that inside cells the RNA is subject to (localized) stabilizing and destabilizing effects that lead to an on average only marginal modulation compared to diluted buffer.


Assuntos
Conformação de Ácido Nucleico , RNA/química , Transferência Ressonante de Energia de Fluorescência , Polietilenoglicóis/química , Polimerização
11.
Chemphyschem ; 16(17): 3681-6, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26420566

RESUMO

Actin polymerization is an essential process in eukaryotic cells that provides a driving force for motility and mechanical resistance for cell shape. By using preformed gelsolin-actin nuclei and applying stopped-flow methodology, we quantitatively studied the elongation kinetics of actin filaments as a function of temperature and pressure in the presence of synthetic and protein crowding agents. We show that the association of actin monomers to the pointed end of double-stranded helical actin filaments (F-actin) proceeds via a transition state that requires an activation energy of 56 kJ mol(-1) for conformational and hydration rearrangements, but exhibits a negligible activation volume, pointing to a compact transition state that is devoid of packing defects. Macromolecular crowding causes acceleration of the F-actin elongation rate and counteracts the deteriorating effect of pressure. The results shed new light on the combined effect of these parameters on the polymerization process of actin, and help us understand the temperature and pressure sensitivity of actin polymerization under extreme conditions.

12.
Phys Chem Chem Phys ; 17(13): 8338-48, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25406896

RESUMO

The cellular environment determines the structure and function of proteins. Marginal changes of the environment can severely affect the energy landscape of protein folding. However, despite the important role of chaperones on protein folding, less is known about chaperonal modulation of protein aggregation and fibrillation considering different classes of chaperones. We find that the pharmacological chaperone O4, the chemical chaperone proline as well as the protein chaperone serum amyloid P component (SAP) are inhibitors of the type 2 diabetes mellitus-related aggregation process of islet amyloid polypeptide (IAPP). By applying biophysical methods such as thioflavin T fluorescence spectroscopy, fluorescence anisotropy, total reflection Fourier-transform infrared spectroscopy, circular dichroism spectroscopy and atomic force microscopy we analyse and compare their inhibition mechanism. We demonstrate that the fibrillation reaction of human IAPP is strongly inhibited by formation of globular, amorphous assemblies by both, the pharmacological and the protein chaperones. We studied the inhibition mechanism under cell-like conditions by using the artificial crowding agents Ficoll 70 and sucrose. Under such conditions the suppressive effect of proline was decreased, whereas the pharmacological chaperone remains active.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Chaperonas Moleculares/metabolismo , Dicroísmo Circular , Dextranos/química , Dextranos/metabolismo , Ficoll/química , Ficoll/metabolismo , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/antagonistas & inibidores , Microscopia de Força Atômica , Chaperonas Moleculares/química , Oxazinas/química , Oxazinas/metabolismo , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Prolina/química , Prolina/metabolismo , Estrutura Secundária de Proteína , Componente Amiloide P Sérico/química , Componente Amiloide P Sérico/metabolismo , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Sacarose/química , Sacarose/metabolismo , Termodinâmica
13.
Angew Chem Int Ed Engl ; 54(8): 2548-51, 2015 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-25557778

RESUMO

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.


Assuntos
Corantes Fluorescentes/química , Polímeros/química , Técnicas Biossensoriais , Fluoresceínas/química , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Polietilenoglicóis/química
14.
Angew Chem Int Ed Engl ; 54(38): 11088-92, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26245168

RESUMO

Biological cells provide a large variety of rodlike filaments, including filamentous actin (F-actin), which can form meshworks and bundles. One key question remaining in the characterization of such network structures revolves around the temperature and pressure stabilities of these architectures as a way to understand why cells actively use proteins for forming them. The packing properties of F-actin in fascin- and Mg(2+) -induced bundles are compared, and significantly different pressure-temperature stabilities are observed because of marked differences in their nature of interaction, solvation, and packing efficiency. Moreover, differences are observed in their morphologies and disintegration scenarios. The pressure-induced dissociation of the actin bundles is reminiscent of a single unbinding transition as observed in other soft elastic manifolds.


Assuntos
Actinas/química , Pressão , Temperatura
15.
J Chromatogr A ; 1730: 465076, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38879975

RESUMO

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

16.
Biophys Chem ; 253: 106226, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31376619

RESUMO

The quaternary structures of insulin glargine and glulisine under formulation conditions and upon dilution using placebo or water were investigated using synchrotron small-angle X-ray scattering. Our results revealed that insulin glulisine in Apidra® is predominantly hexameric in solution with significant fractions of dodecamers and monomers. Upon dilution with placebo, this equilibrium shifts towards monomers. Insulin glargine in Lantus® and Toujeo® is present in a stable hexamer/dimer equilibrium, which is hardly affected by dilution with water down to 1 mg/ml insulin concentration. The results provide exclusive insight into the quaternary structure and thus the association/dissociation properties of the two insulin analogues in marketed formulations.


Assuntos
Hipoglicemiantes/química , Insulina Glargina/química , Insulina/análogos & derivados , Humanos , Insulina/química , Modelos Moleculares , Estrutura Quaternária de Proteína
17.
Sci Rep ; 7(1): 9864, 2017 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-28852183

RESUMO

Guanosine monophosphate, among the nucleotides, has the unique property to self-associate and form nanoscale cylinders consisting of hydrogen-bonded G-quartet disks, which are stacked on top of one another. Such self-assemblies describe not only the basic structural motif of G-quadruplexes formed by, e.g., telomeric DNA sequences, but are also interesting targets for supramolecular chemistry and nanotechnology. The G-quartet stacks serve as an excellent model to understand the fundamentals of their molecular self-association and to unveil their application spectrum. However, the thermodynamic stability of such self-assemblies over an extended temperature and pressure range is largely unexplored. Here, we report a combined FTIR and NMR study on the temperature and pressure stability of G-quartet stacks formed by disodium guanosine 5'-monophosphate (Na25'-GMP). We found that under abyssal conditions, where temperatures as low as 5 °C and pressures up to 1 kbar are reached, the self-association of Na25'-GMP is most favoured. Beyond those conditions, the G-quartet stacks dissociate laterally into monomer stacks without significantly changing the longitudinal dimension. Among the tested alkali cations, K+ is the most efficient one to elevate the temperature as well as the pressure limits of GMP self-assembly.

18.
J Diabetes Res ; 2015: 849017, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26582333

RESUMO

Type 2 diabetes mellitus (T2DM) is an age-related and metabolic disease. Its development is hallmarked, among others, by the dysfunction and degeneration of ß-cells of the pancreatic islets of Langerhans. The major pathological characteristic thereby is the formation of extracellular amyloid deposits consisting of the islet amyloid polypeptide (IAPP). The process of human IAPP (hIAPP) self-association, and the intermediate structures formed as well as the interaction of hIAPP with membrane systems seem to be, at least to a major extent, responsible for the cytotoxicity. Here we present a summary and comparison of the amyloidogenic propensities of hIAPP in bulk solution and in the presence of various neutral and charged lipid bilayer systems as well as biological membranes. We also discuss the cellular effects of macromolecular crowding and osmolytes on the aggregation pathway of hIAPP. Understanding the influence of different cellular factors on hIAPP aggregation will provide more insight into the onset of T2DM and help to develop novel therapeutic strategies.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/metabolismo , Bicamadas Lipídicas/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo
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