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1.
mBio ; 12(2)2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33879585

RESUMO

Convalescent plasma is a promising therapy for coronavirus disease 2019 (COVID-19), but the antibody characteristics that contribute to efficacy remain poorly understood. This study analyzed plasma samples from 126 eligible convalescent blood donors in addition to 15 naive individuals, as well as an additional 20 convalescent individuals as a validation cohort. Multiplexed Fc Array binding assays and functional antibody response assays were utilized to evaluate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody composition and activity. Donor convalescent plasma samples contained a range of antibody cell- and complement-mediated effector functions, indicating the diverse antiviral activity of humoral responses observed among recovered individuals. In addition to viral neutralization, convalescent plasma samples contained antibodies capable of mediating such Fc-dependent functions as complement activation, phagocytosis, and antibody-dependent cellular cytotoxicity against SARS-CoV-2. Plasma samples from a fraction of eligible donors exhibited high activity across all activities evaluated. These polyfunctional plasma samples could be identified with high accuracy with even single Fc Array features, whose correlation with polyfunctional activity was confirmed in the validation cohort. Collectively, these results expand understanding of the diversity of antibody-mediated antiviral functions associated with convalescent plasma, and the polyfunctional antiviral functions suggest that it could retain activity even when its neutralizing capacity is reduced by mutations in variant SARS-CoV-2.IMPORTANCE Convalescent plasma has been deployed globally as a treatment for COVID-19, but efficacy has been mixed. Better understanding of the antibody characteristics that may contribute to its antiviral effects is important for this intervention as well as offer insights into correlates of vaccine-mediated protection. Here, a survey of convalescent plasma activities, including antibody neutralization and diverse effector functions, was used to define plasma samples with broad activity profiles. These polyfunctional plasma samples could be reliably identified in multiple cohorts by multiplex assay, presenting a widely deployable screening test for plasma selection and investigation of vaccine-elicited responses.


Assuntos
Anticorpos Antivirais/sangue , /terapia , /imunologia , Adulto , Idoso , Anticorpos Neutralizantes/sangue , Especificidade de Anticorpos , Citotoxicidade Celular Dependente de Anticorpos , Antígenos Virais/imunologia , Fenômenos Biofísicos , Estudos de Coortes , Ativação do Complemento , Convalescença , Feminino , Humanos , Imunização Passiva , Masculino , Pessoa de Meia-Idade , Fagocitose , Adulto Jovem
2.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806280

RESUMO

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.


Assuntos
Halorrodopsinas/química , Bicamadas Lipídicas/química , Rodopsinas Sensoriais/química , Alcenos/química , Fenômenos Biofísicos , Dimiristoilfosfatidilcolina/química , Espectroscopia de Ressonância de Spin Eletrônica , Halobacteriaceae/química , Halobacteriaceae/efeitos da radiação , Halorrodopsinas/efeitos da radiação , Maleatos/química , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Processos Fotoquímicos , Rodopsinas Sensoriais/efeitos da radiação , Marcadores de Spin
3.
Molecules ; 26(4)2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33669256

RESUMO

O-GlcNAcylation is a posttranslational modification that occurs at serine and threonine residues of protein substrates by the addition of O-linked ß-d-N-acetylglucosamine (GlcNAc) moiety. Two enzymes are involved in this modification: O-GlcNac transferase (OGT), which attaches the GlcNAc residue to the protein substrate, and O-GlcNAcase (OGA), which removes it. This biological balance is important for many biological processes, such as protein expression, cell apoptosis, and regulation of enzyme activity. The extent of this modification has sparked interest in the medical community to explore OGA and OGT as therapeutic targets, particularly in degenerative diseases. While some OGA inhibitors are already in phase 1 clinical trials for the treatment of Alzheimer's disease, OGT inhibitors still have a long way to go. Due to complex expression and instability, the discovery of potent OGT inhibitors is challenging. Over the years, the field has grappled with this problem, and scientists have developed a number of techniques and assays. In this review, we aim to highlight assays and techniques for OGT inhibitor discovery, evaluate their strength for the field, and give us direction for future bioassay methods.


Assuntos
Bioensaio/métodos , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Fenômenos Biofísicos , Química Click , Ligação Proteica
4.
Nat Commun ; 12(1): 1378, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33654081

RESUMO

Glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor-ligand interface and receptor-receptor interface; the unique C-terminal receptor-receptor enables higher order structures on the membrane. Human GITR-GITRL has potential to form a hexameric network of membrane complexes, while murine GITR-GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor-receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.


Assuntos
Proteína Relacionada a TNFR Induzida por Glucocorticoide/química , Fatores de Necrose Tumoral/metabolismo , Animais , Fenômenos Biofísicos , Linhagem Celular , Membrana Celular/metabolismo , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Reprodutibilidade dos Testes , Fatores de Necrose Tumoral/química
5.
Nat Commun ; 12(1): 1548, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750780

RESUMO

Reconstituting artificial proto-cells capable of transducing extracellular signals into cytoskeletal changes can reveal fundamental principles of how non-equilibrium phenomena in cellular signal transduction affect morphogenesis. Here, we generated a Synthetic Morphogenic Membrane System (SynMMS) by encapsulating a dynamic microtubule (MT) aster and a light-inducible signaling system driven by GTP/ATP chemical potential into cell-sized liposomes. Responding to light cues in analogy to morphogens, this biomimetic design embodies basic principles of localized Rho-GTPase signal transduction that generate an intracellular MT-regulator signaling gradient. Light-induced signaling promotes membrane-deforming growth of MT-filaments by dynamically elevating the membrane-proximal tubulin concentration. The resulting membrane deformations enable recursive coupling of the MT-aster with the signaling system, which generates global self-organized morphologies that reorganize towards local external cues in dependence on prior shape. SynMMS thereby signifies a step towards bio-inspired engineering of self-organized cellular morphogenesis.


Assuntos
Sinais (Psicologia) , Lipossomos , Morfogênese/fisiologia , Células Artificiais , Fenômenos Biofísicos , Extensões da Superfície Celular/fisiologia , Centrossomo , Citoesqueleto/metabolismo , Humanos , Lipossomos/química , Microtúbulos/metabolismo , Proteínas Recombinantes , Transdução de Sinais , Estatmina/metabolismo , Biologia Sintética , Tubulina (Proteína)/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
6.
Viruses ; 13(3)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33669141

RESUMO

Viruses are highly dependent on the host they infect. Their dependence triggers processes of virus-host co-adaptation, enabling viruses to explore host resources whilst escaping immunity. Scientists have tackled viral-host interplay at differing levels of complexity-in individual hosts, organs, tissues and cells-and seminal studies advanced our understanding about viral lifecycles, intra- or inter-species transmission, and means to control infections. Recently, it emerged as important to address the physical properties of the materials in biological systems; membrane-bound organelles are only one of many ways to separate molecules from the cellular milieu. By achieving a type of compartmentalization lacking membranes known as biomolecular condensates, biological systems developed alternative mechanisms of controlling reactions. The identification that many biological condensates display liquid properties led to the proposal that liquid-liquid phase separation (LLPS) drives their formation. The concept of LLPS is a paradigm shift in cellular structure and organization. There is an unprecedented momentum to revisit long-standing questions in virology and to explore novel antiviral strategies. In the first part of this review, we focus on the state-of-the-art about biomolecular condensates. In the second part, we capture what is known about RNA virus-phase biology and discuss future perspectives of this emerging field in virology.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Fenômenos Fisiológicos Virais , Animais , Fenômenos Biofísicos , HIV/fisiologia , Humanos , Vírus da Influenza A/fisiologia , Morbillivirus/fisiologia , Organelas/virologia , Vesiculovirus/fisiologia , Viroses/virologia , Internalização do Vírus
8.
Phys Rev Lett ; 126(4): 048101, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33576647

RESUMO

Recent advances in microscopy techniques make it possible to study the growth, dynamics, and response of complex biophysical systems at single-cell resolution, from bacterial communities to tissues and organoids. In contrast to ordered crystals, it is less obvious how one can reliably distinguish two amorphous yet structurally different cellular materials. Here, we introduce a topological earth mover's (TEM) distance between disordered structures that compares local graph neighborhoods of the microscopic cell-centroid networks. Leveraging structural information contained in the neighborhood motif distributions, the TEM metric allows an interpretable reconstruction of equilibrium and nonequilibrium phase spaces and embedded pathways from static system snapshots alone. Applied to cell-resolution imaging data, the framework recovers time ordering without prior knowledge about the underlying dynamics, revealing that fly wing development solves a topological optimal transport problem. Extending our topological analysis to bacterial swarms, we find a universal neighborhood size distribution consistent with a Tracy-Widom law.


Assuntos
Modelos Teóricos , Reconhecimento Automatizado de Padrão/métodos , Algoritmos , Animais , Fenômenos Biofísicos , Coloides/química , Microscopia Crioeletrônica , Drosophila , Entropia , Células Epiteliais/citologia , Interpretação de Imagem Assistida por Computador/métodos , Modelos Biológicos , Modelos Químicos , RNA/química
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 252: 119489, 2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-33524819

RESUMO

Albumin is an attractive protein for the preparation of nanoparticle with possible therapeutic applications, due to its biodegradable, nontoxic, non-immunogenic, and metabolizable properties. Many studies have investigated the formation of albumin nanoparticles, generally by the desolvation or coacervation approaches. One of the most important parameters that should be considered in the formation of nanoparticles is their morphology (size and shape). There are many proposals to control the nanoparticle size, but it remains a challenge for researchers yet. In this study, we showed that control of BSA-based nanoparticles/microparticles size could be achieved by varying the temperature and pH and therefore controlling the rate of aggregation. The aggregation behavior was monitored by UV-Vis spectroscopy, SEM, and dye-binding assay. Our results provide more options for the size and shape control of BSA-based nanoparticle in natural buffer systems. The aggregation of BSA at different temperatures within the range of 50-80 °C were studied under the effect of different pHs in the range of 4.7-6.2. In this research, we found that protein aggregation under extreme conditions of pH and temperature, or at the pH near to pI appears to be amorphous, and at the pH above the pI seems to be the amyloid fibril structure. In some instances where the aggregation is neither too fast nor too slow, in the initial phase of the aggregation process, nanoparticle structures can be identified and separated by mechanistic approaches. This observation suggests that the best condition for monitoring the formation of albumin-based nanoparticles could be pH 5.7, 70 °C. Satisfactory rationalization of all aspects of our experimental observation requires further and more detailed study.


Assuntos
Nanopartículas , Agregados Proteicos , Soroalbumina Bovina , Fenômenos Biofísicos , Concentração de Íons de Hidrogênio
10.
Int J Mol Sci ; 22(4)2021 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-33557020

RESUMO

KIF1A is a kinesin family protein that moves over a long distance along the microtubule (MT) to transport synaptic vesicle precursors in neurons. A single KIF1A molecule can move toward the plus-end of MT in the monomeric form, exhibiting the characteristics of biased Brownian motion. However, how the bias is generated in the Brownian motion of KIF1A has not yet been firmly established. To elucidate this, we conducted a set of molecular dynamics simulations and observed the binding of KIF1A to MT. We found that KIF1A exhibits biased Brownian motion along MT as it binds to MT. Furthermore, we show that the bias toward the plus-end is generated by the ratchet-like energy landscape for the KIF1A-MT interaction, in which the electrostatic interaction and the negatively-charged C-terminal tail (CTT) of tubulin play an essential role. The relevance to the post-translational modifications of CTT is also discussed.


Assuntos
Fenômenos Biofísicos , Cinesina/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios e Motivos de Interação entre Proteínas , Tubulina (Proteína)/química , Algoritmos , Transporte Axonal , Cinesina/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Modelos Teóricos , Neurônios/metabolismo , Ligação Proteica , Tubulina (Proteína)/metabolismo
11.
Nat Commun ; 12(1): 164, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431868

RESUMO

The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.


Assuntos
Canais de Cálcio/metabolismo , Luz , Engenharia de Proteínas , Animais , Fenômenos Biofísicos , Cálcio/metabolismo , Drosophila/metabolismo , Células HEK293 , Células HeLa , Humanos , Degeneração Neural/patologia , Optogenética
12.
Development ; 148(1)2021 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-33408064

RESUMO

Understanding the cellular organization of tissues is key to developmental biology. In order to deal with this complex problem, researchers have taken advantage of reductionist approaches to reveal fundamental morphogenetic mechanisms and quantitative laws. For epithelia, their two-dimensional representation as polygonal tessellations has proved successful for understanding tissue organization. Yet, epithelial tissues bend and fold to shape organs in three dimensions. In this context, epithelial cells are too often simplified as prismatic blocks with a limited plasticity. However, there is increasing evidence that a realistic approach, even from a reductionist perspective, must include apico-basal intercalations (i.e. scutoidal cell shapes) for explaining epithelial organization convincingly. Here, we present an historical perspective about the tissue organization problem. Specifically, we analyze past and recent breakthroughs, and discuss how and why simplified, but realistic, in silico models require scutoidal features to address key morphogenetic events.


Assuntos
Epitélio/anatomia & histologia , Morfogênese , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Forma Celular , Humanos , Modelos Biológicos
14.
Molecules ; 26(3)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499337

RESUMO

During the past decades, solution nuclear magnetic resonance (NMR) spectroscopy has demonstrated itself as a promising tool in drug discovery. Especially, fragment-based drug discovery (FBDD) has benefited a lot from the NMR development. Multiple candidate compounds and FDA-approved drugs derived from FBDD have been developed with the assistance of NMR techniques. NMR has broad applications in different stages of the FBDD process, which includes fragment library construction, hit generation and validation, hit-to-lead optimization and working mechanism elucidation, etc. In this manuscript, we reviewed the current progresses of NMR applications in fragment-based drug discovery, which were illustrated by multiple reported cases. Moreover, the NMR applications in protein-protein interaction (PPI) modulators development and the progress of in-cell NMR for drug discovery were also briefly summarized.


Assuntos
Descoberta de Drogas/métodos , Espectroscopia de Ressonância Magnética/métodos , Animais , Fenômenos Biofísicos , Células/efeitos dos fármacos , Células/metabolismo , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Mapas de Interação de Proteínas , Bibliotecas de Moléculas Pequenas
15.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467625

RESUMO

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.


Assuntos
Regulação Alostérica , Mutação , Dobramento de Proteína , Proteínas/química , Fatores de Transcrição/química , Sítio Alostérico , Animais , Fenômenos Biofísicos , Simulação por Computador , Escherichia coli/metabolismo , Humanos , Ligantes , Espectrometria de Massas , Camundongos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Sialoglicoproteínas/genética , Superóxido Dismutase-1/genética , Termodinâmica , Fatores de Transcrição/genética
16.
Nat Commun ; 12(1): 325, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436562

RESUMO

A crucial step towards engineering biological systems is the ability to precisely tune the genetic response to environmental stimuli. In the case of Escherichia coli inducible promoters, our incomplete understanding of the relationship between sequence composition and gene expression hinders our ability to predictably control transcriptional responses. Here, we profile the expression dynamics of 8269 rationally designed, IPTG-inducible promoters that collectively explore the individual and combinatorial effects of RNA polymerase and LacI repressor binding site strengths. We then fit a statistical mechanics model to measured expression that accurately models gene expression and reveals properties of theoretically optimal inducible promoters. Furthermore, we characterize three alternative promoter architectures and show that repositioning binding sites within promoters influences the types of combinatorial effects observed between promoter elements. In total, this approach enables us to deconstruct relationships between inducible promoter elements and discover practical insights for engineering inducible promoters with desirable characteristics.


Assuntos
Isopropiltiogalactosídeo/farmacologia , Lógica , Regiões Promotoras Genéticas , Sítios de Ligação , Fenômenos Biofísicos , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Fluorescência , Genes Reporter , Mutação/genética , Regiões Operadoras Genéticas/genética , Ligação Proteica , Reprodutibilidade dos Testes , Termodinâmica , Fatores de Transcrição/metabolismo
17.
Commun Biol ; 4(1): 123, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504944

RESUMO

The macro domain is an ADP-ribose (ADPR) binding module, which is considered to act as a sensor to recognize nicotinamide adenine dinucleotide (NAD) metabolites, including poly ADPR (PAR) and other small molecules. The recognition of macro domains with various ligands is important for a variety of biological functions involved in NAD metabolism, including DNA repair, chromatin remodeling, maintenance of genomic stability, and response to viral infection. Nevertheless, how the macro domain binds to moieties with such structural obstacles using a simple cleft remains a puzzle. We systematically investigated the Middle East respiratory syndrome-coronavirus (MERS-CoV) macro domain for its ligand selectivity and binding properties by structural and biophysical approaches. Of interest, NAD, which is considered not to interact with macro domains, was co-crystallized with the MERS-CoV macro domain. Further studies at physiological temperature revealed that NAD has similar binding ability with ADPR because of the accommodation of the thermal-tunable binding pocket. This study provides the biochemical and structural bases of the detailed ligand-binding mode of the MERS-CoV macro domain. In addition, our observation of enhanced binding affinity of the MERS-CoV macro domain to NAD at physiological temperature highlights the need for further study to reveal the biological functions.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , NAD/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Adenosina Difosfato Ribose/metabolismo , Sítios de Ligação , Fenômenos Biofísicos , Cristalização , Cristalografia por Raios X , Humanos , Ligantes , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Poli Adenosina Difosfato Ribose/metabolismo , Ligação Proteica , Domínios Proteicos , Estabilidade Proteica , Termodinâmica
18.
Methods Mol Biol ; 2191: 29-48, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32865737

RESUMO

For a successful characterization of channelrhodopsins with biophysical methods like FTIR, Raman, EPR and NMR spectroscopy and X-ray crystallography, large amounts of purified protein are requested. For proteins of eukaryotic origin, which are poorly expressing in bacterial systems or not at all, the yeast Pichia pastoris represents a promising alternative for overexpression. Here we describe the methods for cloning, overexpression and mutagenesis as well as the purification procedures for channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2), channelrhodopsin-1 from Chlamydomonas augustae (CaChR1) and the scaffold protein MSP1D1 for reconstitution of the membrane proteins into nanodiscs. Finally, protocols are provided to study CaChR1 by FTIR difference spectroscopy and by time-resolved UV/Vis spectroscopy.


Assuntos
Channelrhodopsins/genética , Biologia Molecular/métodos , Nanocompostos/química , Saccharomycetales/genética , Fenômenos Biofísicos , Channelrhodopsins/química , Chlamydomonas/química , Regulação da Expressão Gênica/genética , Luz , Proteínas de Plantas/química , Espectroscopia de Infravermelho com Transformada de Fourier
19.
Methods Mol Biol ; 2247: 155-171, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33301117

RESUMO

Sedimentation velocity analytical ultracentrifugation is a powerful and versatile tool for the characterization of proteins and macromolecular complexes in solution. The direct modeling of the sedimentation process using modern computational strategies allows among others to assess the homogeneity/heterogeneity state of protein samples and to characterize protein associations. In this chapter, we will provide theoretical backgrounds and protocols to analyze the size distribution of protein samples and to determine the affinity of protein-protein hetero-associations.


Assuntos
Fenômenos Biofísicos , Técnicas Biossensoriais , Proteínas/química , Algoritmos , Análise de Dados , Modelos Teóricos , Ligação Proteica , Temperatura , Ultracentrifugação/métodos
20.
Proc Natl Acad Sci U S A ; 118(1)2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-33318128

RESUMO

The sodium pump (Na+, K+-ATPase, NKA) is vital for animal cells, as it actively maintains Na+ and K+ electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF3 - complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K+-antagonism and suggest a route to isoform specific CTSs.


Assuntos
Glicosídeos Cardíacos/química , Glicosídeos Cardíacos/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/química , Sódio/química , Animais , Fenômenos Biofísicos , Digoxina/farmacologia , Modelos Moleculares , Conformação Molecular , Ouabaína/farmacologia , Isoformas de Proteínas , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
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