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1.
Nat Methods ; 20(4): 523-535, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36973549

RESUMEN

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Proteínas , Transferencia Resonante de Energía de Fluorescencia/métodos , Reproducibilidad de los Resultados , Proteínas/química , Conformación Molecular , Laboratorios
2.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artículo en Inglés | MEDLINE | ID: mdl-34845009

RESUMEN

Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding protein-like II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on ∼600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Förster resonance energy transfer and Hydrogen-Deuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.


Asunto(s)
Proteínas/química , Proteínas/metabolismo , Escherichia coli/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Espectrometría de Masas , Modelos Moleculares , Filogenia , Conformación Proteica , Dominios Proteicos , Proteínas/genética
3.
Nano Lett ; 22(21): 8618-8625, 2022 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-36269936

RESUMEN

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.


Asunto(s)
Microscopía , Imagen Individual de Molécula , Microscopía/métodos , Imagen Individual de Molécula/métodos , Colorantes Fluorescentes/química , Algoritmos , Nanotecnología
4.
Angew Chem Int Ed Engl ; 60(51): 26685-26693, 2021 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-34606673

RESUMEN

Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.


Asunto(s)
Carbocianinas/química , Fotones , Transferencia Resonante de Energía de Fluorescencia , Microscopía Fluorescente , Conformación Molecular
5.
Phys Rev Lett ; 125(14): 146001, 2020 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-33064519

RESUMEN

Many processes in chemistry, physics, and biology involve rare events in which the system escapes from a metastable state by surmounting an activation barrier. Examples range from chemical reactions, protein folding, and nucleation events to the catastrophic failure of bridges. A challenge in understanding the underlying mechanisms is that the most interesting information is contained within the rare transition paths, the exceedingly short periods when the barrier is crossed. To establish a model process that enables access to all relevant timescales, although highly disparate, we probe the dynamics of single dielectric particles in a bistable optical trap in solution. Precise localization by high-speed tracking enables us to resolve the transition paths and relate them to the detailed properties of the 3D potential within which the particle diffuses. By varying the barrier height and shape, the experiments provide a stringent benchmark of current theories of transition path dynamics.

6.
J Am Chem Soc ; 139(17): 6062-6065, 2017 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-28394601

RESUMEN

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g., transiently populated intermediates that are inaccessible with existing single-molecule approaches. We demonstrate the potential and reliability of the method on time scales from milliseconds to minutes by investigating the coupled folding and binding reaction of two intrinsically disordered proteins and the conformational changes occurring in a large cytolytic pore-forming toxin.

7.
Angew Chem Int Ed Engl ; 56(25): 7126-7129, 2017 06 12.
Artículo en Inglés | MEDLINE | ID: mdl-28510311

RESUMEN

To enable the investigation of low-affinity biomolecular complexes with confocal single-molecule spectroscopy, we have developed a microfluidic device that allows a concentrated sample to be diluted by up to five orders of magnitude within milliseconds, at the physical limit dictated by diffusion. We demonstrate the capabilities of the device by studying the dissociation kinetics and structural properties of low-affinity protein complexes using single-molecule two-color and three-color Förster resonance energy transfer (FRET). We show that the versatility of the device makes it suitable for studying complexes with dissociation constants from low nanomolar up to 10 µm, thus covering a wide range of biomolecular interactions. The design and precise fabrication of the devices ensure simple yet reliable operation and high reproducibility of the results.


Asunto(s)
Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Imagen Individual de Molécula/métodos , Diseño de Equipo , Transferencia Resonante de Energía de Fluorescencia , Cinética , Reproducibilidad de los Resultados
8.
Biophys J ; 106(2): 440-6, 2014 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-24461019

RESUMEN

Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson's disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15-19 monomers and 34-38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.


Asunto(s)
Dopamina/farmacología , Fotoblanqueo , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/química , Colorantes Fluorescentes/química , Humanos , Estructura Cuaternaria de Proteína
9.
Sci Adv ; 10(39): eado3427, 2024 Sep 27.
Artículo en Inglés | MEDLINE | ID: mdl-39321299

RESUMEN

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is, however, a growing gap between the state of the art and what is accessible to biologists, biochemists, medical researchers, or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed microspectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy, time-correlated single-photon counting and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines, are provided, to allow non-optics specialists to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.


Asunto(s)
Impresión Tridimensional , Imagen Individual de Molécula , Imagen Individual de Molécula/métodos , Microscopía Fluorescente/métodos , Programas Informáticos , Humanos
10.
bioRxiv ; 2024 Jul 10.
Artículo en Inglés | MEDLINE | ID: mdl-38234760

RESUMEN

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and opensource Brick-MIC platform will be a valuable tool for many laboratories worldwide.

11.
Biochemistry ; 51(19): 3960-2, 2012 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-22494024

RESUMEN

α-Synuclein is abundantly present in Lewy bodies, characteristic of Parkinson's disease. Its exact physiological role has yet to be determined, but mitochondrial membrane binding is suspected to be a key aspect of its function. Electron paramagnetic resonance spectroscopy in combination with site-directed spin labeling allowed for a locally resolved analysis of the protein-membrane binding affinity for artificial phospholipid membranes, supported by a study of binding to isolated mitochondria. The data reveal that the binding affinity of the N-terminus is nonuniform.


Asunto(s)
Membrana Celular/metabolismo , alfa-Sinucleína/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Cuerpos de Lewy/metabolismo , Membranas Artificiales , Mutación , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidilgliceroles/química , Fosfatidilgliceroles/metabolismo , Fosfolípidos/química , Fosfolípidos/metabolismo , alfa-Sinucleína/genética
12.
Phys Rev Lett ; 109(20): 203601, 2012 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-23215487

RESUMEN

We have studied the influence of the local density of optical states (LDOS) on the rate and efficiency of Förster resonance energy transfer (FRET) from a donor to an acceptor. The donors and acceptors are dye molecules that are separated by a short strand of double-stranded DNA. The LDOS is controlled by carefully positioning the FRET pairs near a mirror. We find that the energy transfer efficiency changes with LDOS, and that, in agreement with theory, the energy transfer rate is independent of the LDOS, which allows one to quantitatively control FRET systems in a new way. Our results imply a change in the characteristic Förster distance, in contrast to common lore that this distance is fixed for a given FRET pair.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Óptica y Fotónica/métodos , ADN/química , Colorantes Fluorescentes/química , Polimetil Metacrilato/química , Alcohol Polivinílico/química , Termodinámica
13.
Open Biol ; 11(4): 200406, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33823661

RESUMEN

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE-FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE-FRET to monitor protein-protein interactions and conformational states simultaneously.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Fenómenos Químicos , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Análisis Espectral , Relación Estructura-Actividad , Especificidad por Sustrato
15.
Methods Mol Biol ; 1345: 151-69, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26453211

RESUMEN

Amyloid oligomers are considered to be the relevant toxic species in many amyloid diseases and much research effort has been devoted to fully characterize these oligomers. Despite their importance, oligomers have proven to be difficult to characterize structurally. Information on their aggregation number is scarce, largely because standard techniques struggle to provide reliable results. In this chapter, we present two different methods that reproducibly yield fluorescently labeled α-Synuclein oligomers. We then discuss a new approach, combining single-molecule photobleaching and sub-stoichiometric fluorescent labeling, that we have developed to determine the aggregation number of supramolecular protein assemblies.


Asunto(s)
Multimerización de Proteína/genética , Coloración y Etiquetado/métodos , alfa-Sinucleína/química , alfa-Sinucleína/aislamiento & purificación , Colorantes Fluorescentes/química , Humanos , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética
16.
Sci Rep ; 6: 30658, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-27477055

RESUMEN

Although it is well established that the protein α-synuclein (αS) plays an important role in Parkinson's disease, its physiological function remains largely unknown. It has been reported to bind membranes and to play a role in membrane remodeling processes. The mechanism by which αS remodels membranes is still debated; it may either affect its physical properties or act as a chaperone for other membrane associated proteins. To obtain insight into the role of αS in membrane remodeling we investigated the number of αS proteins associated with single small vesicles in a neuronal cell model. Using single-molecule microscopy and photo-bleaching approaches, we most frequently found 70 αS-GFPs per vesicle. Although this number is high enough to modulate physical membrane properties, it is also strikingly similar to the number of synaptobrevins, a putative interaction partner of αS, per vesicle. We therefore hypothesize a dual, synergistic role for αS in membrane remodeling.


Asunto(s)
Membranas/química , Vesículas Sinápticas/química , alfa-Sinucleína/análisis , Animales , Células Cultivadas , Neuronas/química , Ratas Wistar , Imagen Individual de Molécula
17.
Mol Neurobiol ; 47(2): 613-21, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22956232

RESUMEN

In many human diseases, oligomeric species of amyloid proteins may play a pivotal role in cytotoxicity. Many lines of evidence indicate that permeabilization of cellular membranes by amyloid oligomers may be the key factor in disrupting cellular homeostasis. However, the exact mechanisms by which the membrane integrity is impaired remain elusive. One prevailing hypothesis, the so-called amyloid pore hypothesis, assumes that annular oligomeric species embed into lipid bilayers forming transbilayer protein channels. Alternatively, an increased membrane permeability could be caused by thinning of the hydrophobic core of the lipid bilayer due to the incorporation of the oligomers between the tightly packed lipids, which would facilitate the transport of small molecules across the membrane. In this review, we briefly recapitulate our findings on the structure of α-synuclein oligomers and the factors influencing their interaction with lipid bilayers. Our results, combined with work from other groups, suggest that α-synuclein oligomers do not necessarily form pore-like structures. The emerging consensus is that local structural rearrangements of the protein lead to insertion of specific regions into the hydrophobic core of the lipid bilayer, thereby disrupting the lipid packing.


Asunto(s)
Amiloide/química , Membrana Dobles de Lípidos/química , Metabolismo de los Lípidos , alfa-Sinucleína/química , Amiloide/metabolismo , Animales , Humanos , Membrana Dobles de Lípidos/metabolismo , Unión Proteica/fisiología , alfa-Sinucleína/metabolismo
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