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1.
Arch Cardiol Mex ; 2020 Dec 01.
Artículo en Esperanto | MEDLINE | ID: mdl-33262540

RESUMEN

Objetivo: Evaluar el impacto del aislamiento social, preventivo y obligatorio sobre al aumento de peso, el nivel de actividad física, la adherencia al tratamiento y la inmunización antigripal y antineumocócica en pacientes con diabetes tipo 2 (DM2). Método: Se realizó un seguimiento telefónico de pacientes con DM2 luego de al menos 100 días de comienzo del aislamiento. Se compararon dos regiones agrupadas en relación con el número de casos: región 1, más de 150 casos/100, 000 habitantes, y región 2, más de 150 casos/100,000 habitantes. Resultados: Se contactaron 454 pacientes con DM2. El 42% reportaron incremento de peso y el 7% refirió haber aumentado más de 5 kg. En relación con la actividad física, se observó un promedio más bajo en la región 1 (367.5 [0-5698] MET/sem) que en la región 2 (720 [0-7066] MET/sem) (p = 0.0009). La adherencia al tratamiento farmacológico disminuyó en forma global, pero aumentó en las zonas de mayor circulación viral. Por otra parte, hubo un incremento de vacunación antigripal y antineumocócica, alcanzando coberturas del 80% y el 70%, respectivamente. Conclusiones: En una población de alto riesgo cardiovascular y con una prevalencia de sobrepeso/obesidad elevada, el aislamiento social, preventivo y obligatorio se asoció con ganancia de peso y menos actividad física, lo que podría resultar deletéreo sobre la salud cardiovascular de los pacientes con DM2. Por otro lado, se observan algunos aspectos positivos, como el aumento de las inmunizaciones y el incremento de la adherencia en las zonas más afectadas.

2.
Curr Opin Struct Biol ; 66: 129-138, 2020 Nov 24.
Artículo en Inglés | MEDLINE | ID: mdl-33246199

RESUMEN

Evidence regarding protein structure and function manifest the imperative role that dynamics play in proteins, underlining reconsideration of the unanimated sequence-to-structure-to-function paradigm. Structural dynamics portray a heterogeneous energy landscape described by conformational ensembles where each structural representation can be responsible for unique functions or enable macromolecular assemblies. Using the human p27/Cdk2/Cyclin A ternary complex as an example, we highlight the vital role of intramolecular and intermolecular dynamics for target recognition, binding, and inhibition as a critical modulator of cell division. Rapidly sampling configurations is critical for the population of different conformational ensembles encoding functional roles. To garner this knowledge, we present how the integration of (sub)ensemble and single-molecule fluorescence spectroscopy with molecular dynamic simulations can characterize structural dynamics linking the heterogeneous ensembles to function. The incorporation of dynamics into the sequence-to-structure-to-function paradigm promises to assist in tackling various challenges, including understanding the formation and regulation of mesoscale assemblies inside cells.

3.
Nat Commun ; 11(1): 5394, 2020 10 26.
Artículo en Inglés | MEDLINE | ID: mdl-33106483

RESUMEN

FRET experiments can provide state-specific structural information of complex dynamic biomolecular assemblies. However, to overcome the sparsity of FRET experiments, they need to be combined with computer simulations. We introduce a program suite with (i) an automated design tool for FRET experiments, which determines how many and which FRET pairs should be used to minimize the uncertainty and maximize the accuracy of an integrative structure, (ii) an efficient approach for FRET-assisted coarse-grained structural modeling, and all-atom molecular dynamics simulations-based refinement, and (iii) a quantitative quality estimate for judging the accuracy of FRET-derived structures as opposed to precision. We benchmark our tools against simulated and experimental data of proteins with multiple conformational states and demonstrate an accuracy of ~3 Å RMSDCα against X-ray structures for sets of 15 to 23 FRET pairs. Free and open-source software for the introduced workflow is available at https://github.com/Fluorescence-Tools . A web server for FRET-assisted structural modeling of proteins is available at http://nmsim.de .


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Proteínas/química , Automatización , Simulación por Computador , Transferencia Resonante de Energía de Fluorescencia/instrumentación , Modelos Estructurales , Conformación Proteica , Programas Informáticos , Navegador Web
4.
Biophys J ; 119(10): 1929-1936, 2020 Nov 17.
Artículo en Inglés | MEDLINE | ID: mdl-33096078

RESUMEN

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for investigating the structural dynamics of biological macromolecules. smFRET reveals the conformational landscape and dynamic changes of proteins by building on the static structures found using cryo-electron microscopy, x-ray crystallography, and other methods. Combining smFRET with static structures allows for a direct correlation between dynamic conformation and function. Here, we discuss the different experimental setups, fluorescence detection schemes, and data analysis strategies that enable the study of structural dynamics of glutamate signaling across various timescales. We illustrate the versatility of smFRET by highlighting studies of a wide range of questions, including the mechanism of activation and transport, the role of intrinsically disordered segments, and allostery and cooperativity between subunits in biological systems responsible for glutamate signaling.

5.
J Mol Biol ; 432(19): 5411-5429, 2020 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-32735805

RESUMEN

Forkhead box P (FoxP) proteins are unique transcription factors that spatiotemporally regulate gene expression by tethering two chromosome loci together via functional domain-swapped dimers formed through their DNA-binding domains. Further, the differential kinetics on this dimerization mechanism underlie an intricate gene regulation network at physiological conditions. Nonetheless, poor understanding of the structural dynamics and steps of the association process impedes to link the functional domain swapping to human-associated diseases. Here, we have characterized the DNA-binding domain of human FoxP1 by integrating single-molecule Förster resonance energy transfer and hydrogen-deuterium exchange mass spectrometry data with molecular dynamics simulations. Our results confirm the formation of a previously postulated domain-swapped (DS) FoxP1 dimer in solution and reveal the presence of highly populated, heterogeneous, and locally disordered dimeric intermediates along the dimer dissociation pathway. The unique features of FoxP1 provide a glimpse of how intrinsically disordered regions can facilitate domain swapping oligomerization and other tightly regulated association mechanisms relevant in biological processes.

6.
Rev. Soc. Argent. Diabetes ; 54(supl. 2): 71-90, mayo - ago. 2020. graf, tab
Artículo en Español | LILACS, BINACIS | ID: biblio-1122954

RESUMEN

La principal causa de muerte en personas con diabetes mellitus tipo 1 (DM1) es de origen cardiovascular (CV). La duración de la DM1 es uno de los predictores más importantes para infarto agudo de miocardio (IAM), junto con el colesterol de lipoproteínas de baja densidad (cLDL) y HbA1c. El desarrollo de DM1 antes de los 10 años de edad se asocia con un riesgo 90 veces mayor de IAM en mujeres. En la DM1 habría una mayor contribución de un estado de inflamación sistémica de bajo de grado. La combinación de electrocardiograma de ejercicio y una técnica de imagen proporciona valor diagnóstico para la detección de isquemia miocárdica y pronóstico. La evaluación del riesgo CV en el adulto mayor debe ser individualizado y categorizarlo según funcionalidad y comorbilidades a fin de fijar objetivos de control de factores glucémicos y no glucémicos personalizados. Las personas con enfermedad cardíaca conocida o múltiples factores de riesgo cardiovascular (FRCV) deben tener recomendaciones de ejercicio personalizadas; se recomienda el tratamiento intensificado de la glucemia y de los FRCV asociados. En la población pediátrica y adolescentes con DM1 es esencial la detección y tratamiento precoz de los FRCV a fin de prevenir o retrasar el inicio y progresión de eventos CV


The leading cause of death in type 1 diabetes mellitus (T1DM) is cardiovascular. The duration of diabetes is one of the most important predictors for acute myocardial infarction (AMI), along with low-density lipoprotein cholesterol (cLDL) and HbA1c. Being diagnosed of T1DM before age 10 is been associated with a 90 times higher risk of AMI in women. It has been proposed, that a low-grade systemic inflammation state to be great prompt contributor. The combination of exercise electrocardiogram and an imaging technique provides diagnostic value for myocardial ischemia detection and future prognosis. Cardiovascular risk assessment in the older adult should be individualized by categorizing it, according to functionality and patient's co morbidity and customized glycaemic and non-glycaemic targets. People with known heart disease or multiple cardiovascular risk factors should have personalized exercise recommendations, blood glucose intensified treatment and associated risk factors. In paediatric and adolescent population with T1DM, cardiovascular risk factors early screening, recognition and treatment has become essential to prevent or delay the onset and progression of cardiovascular events.


Asunto(s)
Humanos , Diabetes Mellitus Tipo 1 , Terapéutica , Glucemia , Enfermedades Cardiovasculares , Diagnóstico
7.
Nat Commun ; 11(1): 1231, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32144241

RESUMEN

We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.


Asunto(s)
Muramidasa/metabolismo , Proteínas Virales/metabolismo , Bacteriófago T4/enzimología , Bacteriófago T4/genética , Biocatálisis , Cristalografía por Rayos X , Transferencia Resonante de Energía de Fluorescencia , Simulación de Dinámica Molecular , Método de Montecarlo , Muramidasa/química , Muramidasa/genética , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Virales/química , Proteínas Virales/genética
8.
J Mol Biol ; 432(9): 2998-3017, 2020 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-32088186

RESUMEN

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stopped-flow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms.


Asunto(s)
Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Imagen Individual de Molécula/métodos , Sitios de Unión , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/química , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia , Factores Complejos Ternarios/química
9.
Front Biosci (Landmark Ed) ; 25: 43-68, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31585877

RESUMEN

Integrative and hybrid methods have the potential to bridge long-standing knowledge gaps in structural biology. These methods will have a prominent role in the future of the field as we make advances toward a complete, unified representation of biology that spans the molecular and cellular scales. The Department of Physics and Astronomy at Clemson University hosted The Future of Integrative Structural Biology workshop on April 29, 2017 and partially sponsored by partially sponsored by a program of the Oak Ridge Associated Universities (ORAU). The workshop brought experts from multiple structural biology disciplines together to discuss near-term steps toward the goal of a molecular atlas of the cell. The discussion focused on the types of structural data that should be represented, how this data should be represented, and how the time domain might be incorporated into such an atlas. The consensus was that an explorable, map-like Virtual Cell, containing both spatial and temporal data bridging the atomic and cellular length scales obtained by multiple experimental methods, represents the best path toward a complete atlas of the cell.

10.
ACS Appl Mater Interfaces ; 11(51): 48551-48555, 2019 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-31782302

RESUMEN

The photophysics of charge-transfer and recombination mechanisms in a heterojunction structure of CdSe/CdS/Au quantum dots (QDs) are studied by temperature-dependent steady-state photoluminescence (PL) and time-resolved PL (TRPL). We manipulate the charge transfer from core to shell surface by varying the tunneling barrier height resulting from temperature variation and the barrier width resulting from shell thickness variation. The charge-transfer process, which can be described by a tunneling transmission model, is manifested by two competitive recombination processes, an intrinsic exciton emission and a trap emission in the near-infrared (NIR) range. Our study establishes the photophysics foundation for the core/shell/metal application in photocatalyst and optoelectronics.

11.
Nat Commun ; 10(1): 1676, 2019 04 11.
Artículo en Inglés | MEDLINE | ID: mdl-30976006

RESUMEN

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease.


Asunto(s)
División Celular/fisiología , Ciclina A/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Cristalografía por Rayos X , Ciclina A/aislamiento & purificación , Quinasa 2 Dependiente de la Ciclina/aislamiento & purificación , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/aislamiento & purificación , Proteínas de Fusión bcr-abl/metabolismo , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Fosforilación/fisiología , Unión Proteica/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Estructura Terciaria de Proteína/fisiología , Proteolisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Treonina/metabolismo , Tirosina/metabolismo , Familia-src Quinasas/aislamiento & purificación , Familia-src Quinasas/metabolismo
12.
Biomolecules ; 9(3)2019 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-30909517

RESUMEN

The common conception of intrinsically disordered proteins (IDPs) is that they stochastically sample all possible configurations driven by thermal fluctuations. This is certainly true for many IDPs, which behave as swollen random coils that can be described using polymer models developed for homopolymers. However, the variability in interaction energy between different amino acid sequences provides the possibility that some configurations may be strongly preferred while others are forbidden. In compact globular IDPs, core hydration and packing density can vary between segments of the polypeptide chain leading to complex conformational dynamics. Here, we describe a growing number of proteins that appear intrinsically disordered by biochemical and bioinformatic characterization but switch between restricted regions of conformational space. In some cases, spontaneous switching between conformational ensembles was directly observed, but few methods can identify when an IDP is acting as a restricted chain. Such switching between disparate corners of conformational space could bias ligand binding and regulate the volume of IDPs acting as structural or entropic elements. Thus, mapping the accessible energy landscape and capturing dynamics across a wide range of timescales are essential to recognize when an IDP is acting as such a switch.


Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Humanos , Conformación Proteica
14.
Nat Methods ; 15(9): 669-676, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-30171252

RESUMEN

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Laboratorios/normas , Reproducibilidad de los Resultados
15.
Nat Commun ; 9(1): 3724, 2018 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-30214057

RESUMEN

Previous studies of the N-terminal PDZ tandem from PSD-95 produced divergent models and failed to identify interdomain contacts stabilizing the structure. We used ensemble and single-molecule FRET along with replica-exchange molecular dynamics to fully characterize the energy landscape. Simulations and experiments identified two conformations: an open-like conformation with a small contact interface stabilized by salt bridges, and a closed-like conformation with a larger contact interface stabilized by surface-exposed hydrophobic residues. Both interfaces were confirmed experimentally. Proximity of interdomain contacts to the binding pockets may explain the observed coupling between conformation and binding. The low-energy barrier between conformations allows submillisecond dynamics, which were time-averaged in previous NMR and FRET studies. Moreover, the small contact interfaces were likely overridden by lattice contacts as crystal structures were rarely sampled in simulations. Our hybrid approach can identify transient interdomain interactions, which are abundant in multidomain proteins yet often obscured by dynamic averaging.


Asunto(s)
Homólogo 4 de la Proteína Discs Large/química , Dominios PDZ , Animales , Simulación por Computador , Disulfuros , Escherichia coli/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Fotones , Unión Proteica , Ratas , Factores de Transcripción
16.
Medicina (B Aires) ; 78(3): 185-193, 2018.
Artículo en Español | MEDLINE | ID: mdl-29940545

RESUMEN

Diabetes mellitus is currently a serious public health problem worldwide, that increases the risk of presenting microvascular and macrovascular complications. Although achieving the recommended blood glucose goals reduces the risk of microvascular complications, the effect of the drugs used to treat hyperglycemia on macrovascular complications and cardiovascular death is a cause for concern. In this context, the regulatory agencies have modified the regulations for the approval of new drugs in diabetes, by adding the need to demonstrate that they are capable of lowering blood glucose levels together with a solid assessment of cardiovascular safety. The objective of this study is to review the cardiovascular effects of the new families of non-insulin drugs, with special emphasis on their effect on the risk of major cardiovascular events. In recent years, it has finally been confirmed that some of the drugs used to treat diabetes are not only safe from a cardiovascular point of view, but have even shown capacity to reduce the risk of cardiovascular disease in type 2 diabetes mellitus. The evidence obtained determined the updating of some current therapeutic guidelines when cardiovascular risk should be considered a fundamental variable at the time of therapeutic choice in patients with diabetes.


Asunto(s)
Enfermedades Cardiovasculares/inducido químicamente , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/efectos adversos , Humanos , Hipoglucemiantes/uso terapéutico , Factores de Riesgo
17.
Medicina (B.Aires) ; 78(3): 185-193, jun. 2018. ilus, tab
Artículo en Español | LILACS-Express | LILACS | ID: biblio-954975

RESUMEN

La diabetes mellitus constituye actualmente un grave problema de salud pública a nivel mundial, que incrementa el riesgo de presentar complicaciones tanto microvasculares como macrovasculares. Aunque lograr los objetivos de glucemia recomendados reduce el riesgo de complicaciones microvasculares, el efecto de los fármacos para tratar la hiperglucemia sobre las complicaciones macrovasculares y la muerte cardiovascular es motivo de preocupación. En este contexto, las agencias regulatorias han modificado la normativa para la aprobación de nuevos fármacos en diabetes, de forma que establecen la necesidad de demostrar que son capaces de disminuir la glucemia junto con una evaluación sólida de la seguridad cardiovascular. El objetivo de este trabajo es revisar los efectos cardiovasculares de las nuevas familias de fármacos no insulínicos, en especial en su efecto sobre el riesgo de eventos cardiovasculares mayores. En los últimos años, finalmente, se ha confirmado que algunos fármacos para tratar la diabetes no solo son seguros desde el punto de vista cardiovascular, sino que incluso han mostrado capacidad para reducir el riesgo de enfermedad cardiovascular en la diabetes mellitus tipo 2. La evidencia obtenida ha determinado la actualización de algunas guías terapéuticas vigentes cuando el riesgo cardiovascular debería considerarse una variable fundamental al momento de la elección terapéutica en pacientes con diabetes.


Diabetes mellitus is currently a serious public health problem worldwide, that increases the risk of presenting microvascular and macrovascular complications. Although achieving the recommended blood glucose goals reduces the risk of microvascular complications, the effect of the drugs used to treat hyperglycemia on macrovascular complications and cardiovascular death is a cause for concern. In this context, the regulatory agencies have modified the regulations for the approval of new drugs in diabetes, by adding the need to demonstrate that they are capable of lowering blood glucose levels together with a solid assessment of cardiovascular safety. The objective of this study is to review the cardiovascular effects of the new families of non-insulin drugs, with special emphasis on their effect on the risk of major cardiovascular events. In recent years, it has finally been confirmed that some of the drugs used to treat diabetes are not only safe from a cardiovascular point of view, but have even shown capacity to reduce the risk of cardiovascular disease in type 2 diabetes mellitus. The evidence obtained determined the updating of some current therapeutic guidelines when cardiovascular risk should be considered a fundamental variable at the time of therapeutic choice in patients with diabetes.

18.
J Vis Exp ; (123)2017 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-28570518

RESUMEN

A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores de N-Metil-D-Aspartato/química , Fluorescencia , Ligandos , Conformación Molecular , Unión Proteica , Dominios Proteicos , Receptores de N-Metil-D-Aspartato/metabolismo
19.
Biochemistry ; 56(15): 2149-2160, 2017 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-28318265

RESUMEN

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is highly abundant in neurons, where its concentration reaches that typically found for cytoskeletal proteins. Functional reasons for such a high concentration are not known, but given the multitude of known binding partners for CaMKII, a role as a scaffolding molecule has been proposed. In this report, we provide experimental evidence that demonstrates a novel structural role for CaMKII. We discovered that CaMKII forms filaments that can extend for several micrometers in the presence of certain divalent cations (Zn2+, Cd2+, and Cu2+) but not with others (Ca2+, Mg2+, Co2+, and Ni2+). Once formed, depleting the divalent ion concentration with chelators completely dissociated the filaments, and this process could be repeated by cyclic addition and removal of divalent ions. Using the crystal structure of the CaMKII holoenzyme, we computed an electrostatic potential map of the dodecameric complex to predict divalent ion binding sites. This analysis revealed a potential surface-exposed divalent ion binding site involving amino acids that also participate in calmodulin (CaM) binding and suggested CaM binding might inhibit formation of the filaments. As predicted, Ca2+/CaM binding both inhibited divalent ion-induced filament formation and could disassemble preformed filaments. Interestingly, CaMKII within the filaments retains the capacity to autophosphorylate; however, activity toward exogenous substrates is significantly decreased. Activity is restored upon filament disassembly. We compile our results with structural and mechanistic data from the literature to propose a model of Zn2+-mediated CaMKII filament formation, in which assembly and activity are further regulated by Ca2+/CaM.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Citoesqueleto/química , Zinc/química , Animales , Línea Celular , Cinética , Fosforilación , Ratas , Spodoptera , Electricidad Estática
20.
J Biol Chem ; 291(31): 16175-85, 2016 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-27226581

RESUMEN

The N-methyl-d-aspartate (NMDA) receptors are heteromeric non-selective cation channels that require the binding of glycine and glutamate for gating. Based on crystal structures, the mechanism of partial agonism at the glycine-binding site is thought to be mediated by a shift in the conformational equilibrium between an open clamshell and a closed clamshell-like structure of the bilobed ligand-binding domain (LBD). Using single-molecule Förster resonance energy transfer (smFRET) and multiparameter fluorescence detection, which allows us to study the conformational states and dynamics in the submillisecond time scale, we show that there are at least three conformational states explored by the LBD: the low FRET, medium FRET, and high FRET states. The distance of the medium and low FRET states corresponds to what has been observed in crystallography structures. We show that the high FRET state, which would represent a more closed clamshell conformation than that observed in the crystal structure, is most likely the state initiating activation, as evidenced by the fact that the fraction of the protein in this state correlates well with the extent of activation. Furthermore, full agonist bound LBDs show faster dynamic motions between the medium and high FRET states, whereas they show slower dynamics when bound to weaker agonists or to antagonists.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Receptores de N-Metil-D-Aspartato/química , Animales , Línea Celular , Humanos , Dominios Proteicos , Ratas
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