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1.
Proc Natl Acad Sci U S A ; 121(31): e2220020121, 2024 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-39042693

RESUMEN

Expansion of intronic GGGGCC repeats in the C9orf72 gene causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Transcription of the expanded repeats results in the formation of RNA-containing nuclear foci and altered RNA metabolism. In addition, repeat-associated non-AUG (RAN) translation of the expanded GGGGCC-repeat sequence results in the production of highly toxic dipeptide-repeat (DPR) proteins. GGGGCC repeat-containing transcripts form G-quadruplexes, which are associated with formation of RNA foci and RAN translation. Zfp106, an RNA-binding protein essential for motor neuron survival in mice, suppresses neurotoxicity in a Drosophila model of C9orf72 ALS. Here, we show that Zfp106 inhibits formation of RNA foci and significantly reduces RAN translation caused by GGGGCC repeats in cultured mammalian cells, and we demonstrate that Zfp106 coexpression reduces the levels of DPRs in C9orf72 patient-derived cells. Further, we show that Zfp106 binds to RNA G-quadruplexes and causes a conformational change in the G-quadruplex structure formed by GGGGCC repeats. Together, these data demonstrate that Zfp106 suppresses the formation of RNA foci and DPRs caused by GGGGCC repeats and suggest that the G-quadruplex RNA-binding function of Zfp106 contributes to its suppression of GGGGCC repeat-mediated cytotoxicity.


Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72 , G-Cuádruplex , Proteínas de Unión al ARN , ARN , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Unión Proteica , Biosíntesis de Proteínas , ARN/metabolismo , ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética
2.
Nat Chem Biol ; 20(6): 751-760, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38480980

RESUMEN

Transmembrane (TM) domains as simple as a single span can perform complex biological functions using entirely lipid-embedded chemical features. Computational design has the potential to generate custom tool molecules directly targeting membrane proteins at their functional TM regions. Thus far, designed TM domain-targeting agents have been limited to mimicking the binding modes and motifs of natural TM interaction partners. Here, we demonstrate the design of de novo TM proteins targeting the erythropoietin receptor (EpoR) TM domain in a custom binding topology competitive with receptor homodimerization. The TM proteins expressed in mammalian cells complex with EpoR and inhibit erythropoietin-induced cell proliferation. In vitro, the synthetic TM domain complex outcompetes EpoR homodimerization. Structural characterization reveals that the complex involves the intended amino acids and agrees with our designed molecular model of antiparallel TM helices at 1:1 stoichiometry. Thus, membrane protein TM regions can now be targeted in custom-designed topologies.


Asunto(s)
Proteínas de la Membrana , Unión Proteica , Receptores de Eritropoyetina , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Receptores de Eritropoyetina/metabolismo , Receptores de Eritropoyetina/química , Modelos Moleculares , Proliferación Celular/efectos de los fármacos , Receptores de Citocinas/metabolismo , Receptores de Citocinas/química , Secuencia de Aminoácidos , Multimerización de Proteína , Animales , Células HEK293
3.
Faraday Discuss ; 232(0): 9-48, 2021 12 24.
Artículo en Inglés | MEDLINE | ID: mdl-34693965

RESUMEN

Membrane-peptide interactions play critical roles in many cellular and organismic functions, including protection from infection, remodeling of membranes, signaling, and ion transport. Peptides interact with membranes in a variety of ways: some associate with membrane surfaces in either intrinsically disordered conformations or well-defined secondary structures. Peptides with sufficient hydrophobicity can also insert vertically as transmembrane monomers, and many associate further into membrane-spanning helical bundles. Indeed, some peptides progress through each of these stages in the process of forming oligomeric bundles. In each case, the structure of the peptide and the membrane represent a delicate balance between peptide-membrane and peptide-peptide interactions. We will review this literature from the perspective of several biologically important systems, including antimicrobial peptides and their mimics, α-synuclein, receptor tyrosine kinases, and ion channels. We also discuss the use of de novo design to construct models to test our understanding of the underlying principles and to provide useful leads for pharmaceutical intervention of diseases.


Asunto(s)
Péptidos , Interacciones Hidrofóbicas e Hidrofílicas , Estructura Secundaria de Proteína
4.
Nat Chem Biol ; 14(9): 870-875, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30061717

RESUMEN

Amyloids adopt 'cross-ß' structures composed of long, twisted fibrils with ß-strands running perpendicular to the fibril axis. Recently, a toxic peptide was proposed to form amyloid-like cross-α structures in solution, with a planar bilayer-like assembly observed in the crystal structure. Here we crystallographically characterize designed peptides that assemble into spiraling cross-α amyloid-like structures, which resemble twisted ß-amyloid fibrils. The peptides form helical dimers, stabilized by packing of small and apolar residues, and the dimers further assemble into cross-α amyloid-like fibrils with superhelical pitches ranging from 170 Å to 200 Å. When a small residue that appeared critical for packing was converted to leucine, it resulted in structural rearrangement to a helical polymer. Fluorescently tagged versions of the designed peptides form puncta in mammalian cells, which recover from photobleaching with markedly different kinetics. These structural folds could be potentially useful for directing in vivo protein assemblies with predetermined spacing and stabilities.


Asunto(s)
Amiloide/química , Péptidos/química , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Péptidos/síntesis química , Conformación Proteica
5.
J Am Chem Soc ; 141(18): 7320-7326, 2019 05 08.
Artículo en Inglés | MEDLINE | ID: mdl-30998340

RESUMEN

Infrared (IR) spectroscopy has provided considerable insight into the structures, dynamics, and formation mechanisms of amyloid fibrils. IR probes, such as main chain 13C═18O, have been widely employed to obtain site-specific structural information, yet only secondary structures and strand-to-strand arrangements can be probed. Very few nonperturbative IR probes are available to report on the side-chain conformation and environments, which are critical to determining sheet-to-sheet arrangements in steric zippers within amyloids. Polar residues, such as glutamine, contribute significantly to the stability of amyloids and thus are frequently found in core regions of amyloid peptides/proteins. Furthermore, polyglutamine (polyQ) repeats form toxic aggregates in several neurodegenerative diseases. Here we report the synthesis and application of a new nonperturbative IR probe-glutamine side chain 13C═18O. We use side chain 13C═18O labeling and isotope dilution to detect the presence of intermolecularly hydrogen-bonded arrays of glutamine side chains (Gln ladders) in amyloid-forming peptides. Moreover, the line width of the 13C═18O peak is highly sensitive to its local hydration environment. The IR data from side chain labeling allows us to unambiguously determine the sheet-to-sheet arrangement in a short amyloid-forming peptide, GNNQQNY, providing insight that was otherwise inaccessible through main chain labeling. With several different fibril samples, we also show the versatility of this IR probe in studying the structures and aggregation kinetics of amyloids. Finally, we demonstrate the capability of modeling amyloid structures with IR data using the integrative modeling platform (IMP) and the potential of integrating IR with other biophysical methods for more accurate structural modeling. Together, we believe that side chain 13C═18O will complement main chain isotope labeling in future IR studies of amyloids and integrative modeling using IR data will significantly expand the power of IR spectroscopy to elucidate amyloid assemblies.


Asunto(s)
Amiloide/síntesis química , Glutamina/química , Marcaje Isotópico , Sondas Moleculares/química , Amiloide/química , Espectrofotometría Infrarroja
6.
J Am Chem Soc ; 139(26): 8837-8845, 2017 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-28472884

RESUMEN

The interplay between the intracellular gate and the selectivity filter underlies the structural basis for gating in potassium ion channels. Using a combination of protein semisynthesis, two-dimensional infrared (2D IR) spectroscopy, and molecular dynamics (MD) simulations, we probe the ion occupancy at the S1 binding site in the constricted state of the selectivity filter of the KcsA channel when the intracellular gate is open and closed. The 2D IR spectra resolve two features, whose relative intensities depend on the state of the intracellular gate. By matching the experiment to calculated 2D IR spectra of structures predicted by MD simulations, we identify the two features as corresponding to states with S1 occupied or unoccupied by K+. We learn that S1 is >70% occupied when the intracellular gate is closed and <15% occupied when the gate is open. Comparison of MD trajectories show that opening of the intracellular gate causes a structural change in the selectivity filter, which leads to a change in the ion occupancy. This work reveals the complexity of the conformational landscape of the K+ channel selectivity filter and its dependence on the state of the intracellular gate.


Asunto(s)
Activación del Canal Iónico , Simulación de Dinámica Molecular , Canales de Potasio/química , Sitios de Unión , Espectrofotometría Infrarroja
7.
J Chem Phys ; 143(10): 104201, 2015 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-26374025

RESUMEN

Determining the number of molecules in a given assembly, such as the number of proteins in a toxic aggregate, is often critical to understanding chemistry and function. Herein, we report a variation of a limitless method for counting photoactivatable fluorescent dyes in which single dye molecules are photoswitched to a fluorescent state, counted, and then irreversibly photobleached. We use this method to count the number of CAGE 552 covalently bound to the surface of 500 nm polystyrene beads. Activation of CAGE 552 was achieved with a 405 nm laser pulse. Once activated, the dye was excited with 532 nm light, and the fluorescence emission was collected with a CCD camera. The results from the fluorescence experiments were then compared to bulk fluorescence measurements to assess the error in counting. There are other ways of counting molecules, such as photobleaching and statistical analysis of reversible switchable chromophores. The method reported here provides a lower bound to the number of chromophores, with no upper limit to the number of molecules that can be quantified.


Asunto(s)
Colorantes Fluorescentes/química , Colorantes Fluorescentes/efectos de la radiación , Microscopía Fluorescente/métodos , Imagen Óptica/métodos , Fluorescencia , Procesos Fotoquímicos , Poliestirenos
8.
Nat Chem ; 15(7): 1012-1021, 2023 07.
Artículo en Inglés | MEDLINE | ID: mdl-37308712

RESUMEN

Selective proton transport through proteins is essential for forming and using proton gradients in cells. Protons are conducted along hydrogen-bonded 'wires' of water molecules and polar side chains, which, somewhat surprisingly, are often interrupted by dry apolar stretches in the conduction pathways, inferred from static protein structures. Here we hypothesize that protons are conducted through such dry spots by forming transient water wires, often highly correlated with the presence of the excess protons in the water wire. To test this hypothesis, we performed molecular dynamics simulations to design transmembrane channels with stable water pockets interspersed by apolar segments capable of forming flickering water wires. The minimalist designed channels conduct protons at rates similar to viral proton channels, and they are at least 106-fold more selective for H+ over Na+. These studies inform the mechanisms of biological proton conduction and the principles for engineering proton-conductive materials.


Asunto(s)
Protones , Agua , Agua/química , Proteínas/química , Hidrógeno , Simulación de Dinámica Molecular
9.
Nat Chem ; 13(7): 643-650, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33972753

RESUMEN

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.


Asunto(s)
Canales Iónicos/química , Membrana Dobles de Lípidos/química , Péptidos/química , Secuencia de Aminoácidos , Simulación de Dinámica Molecular , Conformación Proteica en Hélice alfa , Ingeniería de Proteínas , Solubilidad , Agua/química
10.
Sci Adv ; 7(16)2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33853786

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate-ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.


Asunto(s)
Dominio Catalítico/fisiología , Unión Proteica/fisiología , Proteínas no Estructurales Virales/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Proteínas no Estructurales Virales/genética , Tratamiento Farmacológico de COVID-19
11.
bioRxiv ; 2021 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-34013269

RESUMEN

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

12.
Res Sq ; 2021 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-34031651

RESUMEN

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

13.
bioRxiv ; 2020 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-33269349

RESUMEN

The SARS-CoV-2 macrodomain (Mac1) within the non-structural protein 3 (Nsp3) counteracts host-mediated antiviral ADP-ribosylation signalling. This enzyme is a promising antiviral target because catalytic mutations render viruses non-pathogenic. Here, we report a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of diverse fragment libraries resulted in 214 unique macrodomain-binding fragments, out of 2,683 screened. An additional 60 molecules were selected from docking over 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several crystallographic and docking fragment hits were validated for solution binding using three biophysical techniques (DSF, HTRF, ITC). Overall, the 234 fragment structures presented explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macrodomain inhibitors.

14.
Science ; 370(6523): 1473-1479, 2020 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-33154106

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.


Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/inmunología , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Afinidad de Anticuerpos , Chlorocebus aethiops , Microscopía por Crioelectrón , Humanos , Pruebas de Neutralización , Unión Proteica , Estabilidad Proteica , Anticuerpos de Dominio Único/química , Glicoproteína de la Espiga del Coronavirus/química , Células Vero
15.
bioRxiv ; 2020 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-32817938

RESUMEN

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

16.
Chem Commun (Camb) ; 55(35): 5095-5098, 2019 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-30957824

RESUMEN

Recently, l-4-cyanotryptophan has been shown to be an efficient blue fluorescence emitter, with the potential to enable novel applications in biological spectroscopy and microscopy. However, lack of facile synthetic routes to this unnatural amino acid limits its wide use. Herein, we describe an expedient approach to synthesize Fmoc protected l-4-cyanotryptophan with high optical purity (>99%). Additionally, we test the utility of this blue fluorophore in imaging cell-membrane-bound peptides and in determining peptide-membrane binding constants.


Asunto(s)
Colorantes Fluorescentes/química , Triptófano/análogos & derivados , Triptófano/química , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/síntesis química , Células HeLa , Humanos , Proteínas de la Membrana/síntesis química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente/métodos , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Unión Proteica , Triptófano/síntesis química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo
17.
J Phys Chem B ; 121(10): 2331-2338, 2017 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-28225620

RESUMEN

The physical origins of vibrational frequency shifts have been extensively studied in order to understand noncovalent intermolecular interactions in the condensed phase. In the case of carbonyls, vibrational solvatochromism, MD simulations, and vibrational Stark spectroscopy suggest that the frequency shifts observed in simple solvents arise predominately from the environment's electric field due to the vibrational Stark effect. This is contrary to many previously invoked descriptions of vibrational frequency shifts, such as bond polarization, whereby the bond's force constant and/or partial nuclear charges are altered due to the environment, often illustrated in terms of favored resonance structures. Here we test these hypotheses using vibrational solvatochromism as measured using 2D IR to assess the solvent dependence of the bond anharmonicity. These results indicate that the carbonyl bond's anharmonicity is independent of solvent as tested using hexanes, DMSO, and D2O and is supported by simulated 2D spectra. In support of the linear vibrational Stark effect, these 2D IR measurements are consistent with the assertion that the Stark tuning rate is unperturbed by the electric field generated by both hydrogen and non-hydrogen bonding environments and further extends the general applicability of carbonyl probes for studying intermolecular interactions.


Asunto(s)
Acetofenonas/química , Solventes/química , Óxido de Deuterio/química , Dimetilsulfóxido/química , Hexanos/química , Enlace de Hidrógeno , Espectrofotometría Infrarroja , Electricidad Estática , Vibración
18.
Science ; 353(6303): 1040-1044, 2016 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-27701114

RESUMEN

Potassium channels are responsible for the selective permeation of K+ ions across cell membranes. K+ ions permeate in single file through the selectivity filter, a narrow pore lined by backbone carbonyls that compose four K+ binding sites. Here, we report on the two-dimensional infrared (2D IR) spectra of a semisynthetic KcsA channel with site-specific heavy (13C18O) isotope labels in the selectivity filter. The ultrafast time resolution of 2D IR spectroscopy provides an instantaneous snapshot of the multi-ion configurations and structural distributions that occur spontaneously in the filter. Two elongated features are resolved, revealing the statistical weighting of two structural conformations. The spectra are reproduced by molecular dynamics simulations of structures with water separating two K+ ions in the binding sites, ruling out configurations with ions occupying adjacent sites.


Asunto(s)
Proteínas Bacterianas/química , Modelos Químicos , Canales de Potasio/química , Proteínas Bacterianas/síntesis química , Sitios de Unión , Isótopos de Carbono/química , Cristalografía por Rayos X , Marcaje Isotópico , Simulación de Dinámica Molecular , Isótopos de Oxígeno/química , Canales de Potasio/síntesis química , Conformación Proteica , Sodio/química , Espectrofotometría Infrarroja , Agua/química
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