RESUMO
We develop magnetic cytoskeleton affinity (MiCA) purification, which allows for rapid isolation of molecular motors conjugated to large multivalent quantum dots, in miniscule quantities, which is especially useful for single-molecule applications. When purifying labeled molecular motors, an excess of fluorophores or labels is usually used. However, large labels tend to sediment during the centrifugation step of microtubule affinity purification, a traditionally powerful technique for motor purification. This is solved with MiCA, and purification time is cut from 2 h to 20 min, a significant time-savings when it needs to be done daily. For kinesin, MiCA works with as little as 0.6 µg protein, with yield of â¼27%, compared to 41% with traditional purification. We show the utility of MiCA purification in a force-gliding assay with kinesin, allowing, for the first time, simultaneous determination of whether the force from each motor in a multiple-motor system drives or hinders microtubule movement. Furthermore, we demonstrate rapid purification of just 30 ng dynein-dynactin-BICD2N-QD (DDB-QD), ordinarily a difficult protein-complex to purify.
Assuntos
Citoesqueleto/química , Microtúbulos/química , Proteínas Motores Moleculares/química , Pontos Quânticos/química , Animais , Cromatografia de Afinidade , Complexo Dinactina/isolamento & purificação , Dineínas/isolamento & purificação , Humanos , Proteínas Motores Moleculares/isolamento & purificação , Coloração e Rotulagem , Fatores de TempoRESUMO
According to L-edge sum rules, the number of 3d vacancies at a transition metal site is directly proportional to the integrated intensity of the L-edge X-ray absorption spectrum (XAS) for the corresponding metal complex. In this study, the numbers of 3d holes are characterized quantitatively or semi-quantitatively for a series of manganese (Mn) and nickel (Ni) complexes, including the electron configurations 3d10â 3d0. In addition, extremely dilute (<0.1% wt/wt) Ni enzymes were examined by two different approaches: (1) by using a high resolution superconducting tunnel junction X-ray detector to obtain XAS spectra with a very high signal-to-noise ratio, especially in the non-variant edge jump region; and (2) by adding an inert tracer to the sample that provides a prominent spectral feature to replace the weak edge jump for intensity normalization. In this publication, we present for the first time: (1) L-edge sum rule analysis for a series of Mn and Ni complexes that include electron configurations from an open shell 3d0 to a closed shell 3d10; (2) a systematic analysis on the uncertainties, especially on that from the edge jump, which was missing in all previous reports; (3) a clearly-resolved edge jump between pre-L3 and post-L2 regions from an extremely dilute sample; (4) an evaluation of an alternative normalization standard for L-edge sum rule analysis. XAS from two copper (Cu) proteins measured using a conventional semiconductor X-ray detector are also repeated as bridges between Ni complexes and dilute Ni enzymes. The differences between measuring 1% Cu enzymes and measuring <0.1% Ni enzymes are compared and discussed. This study extends L-edge sum rule analysis to virtually any 3d metal complex and any dilute biological samples that contain 3d metals.
RESUMO
Quantum dots are fluorescent nanoparticles used to detect and image proteins and nucleic acids. Compared with organic dyes and fluorescent proteins, these nanocrystals have enhanced brightness, photostability, and wavelength tunability, but their larger size limits their use. Recently, multidentate polymer coatings have yielded stable quantum dots with small hydrodynamic dimensions (≤10 nm) due to high-affinity, compact wrapping around the nanocrystal. However, this coating technology has not been widely adopted because the resulting particles are frequently heterogeneous and clustered, and conjugation to biological molecules is difficult to control. In this article we develop new polymeric ligands and optimize coating and bioconjugation methodologies for core/shell CdSe/Cd(x)Zn(1-x)S quantum dots to generate homogeneous and compact products. We demonstrate that "ligand stripping" to rapidly displace nonpolar ligands with hydroxide ions allows homogeneous assembly with multidentate polymers at high temperature. The resulting aqueous nanocrystals are 7-12 nm in hydrodynamic diameter, have quantum yields similar to those in organic solvents, and strongly resist nonspecific interactions due to short oligoethylene glycol surfaces. Compared with a host of other methods, this technique is superior for eliminating small aggregates identified through chromatographic and single-molecule analysis. We also demonstrate high-efficiency bioconjugation through azide-alkyne click chemistry and self-assembly with hexa-histidine-tagged proteins that eliminate the need for product purification. The conjugates retain specificity of the attached biomolecules and are exceptional probes for immunofluorescence and single-molecule dynamic imaging. These results are expected to enable broad utilization of compact, biofunctional quantum dots for studying crowded macromolecular environments such as the neuronal synapse and cellular cytoplasm.
Assuntos
Acrilatos/química , Resinas Acrílicas/química , Técnicas Biossensoriais/métodos , Pontos Quânticos/química , Succinimidas/química , Compostos de Cádmio/química , DNA/química , Receptores ErbB/química , Humanos , Imunoconjugados/química , Ligantes , Compostos de Selênio/químicaRESUMO
Immunofluorescence, a powerful technique to detect specific targets using fluorescently labeled antibodies, has been widely used in both scientific research and clinical diagnostics. The probes should be made with small antibodies and high brightness. We conjugated GFP binding protein (GBP) nanobodies, small single-chain antibodies from llamas, with new â¼7 nm quantum dots. These provide simple and versatile immunofluorescence nanoprobes with nanometer accuracy and resolution. Using the new probes we tracked the walking of individual kinesin motors and measured their 8 nm step sizes; we tracked Piezo1 channels, which are eukaryotic mechanosensitive channels; we also tracked AMPA receptors on living neurons. Finally, we used a new super-resolution algorithm based on blinking of (small) quantum dots that allowed â¼2 nm precision.
Assuntos
Microscopia de Fluorescência/métodos , Pontos Quânticos/química , Anticorpos de Domínio Único/química , Algoritmos , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Canais Iônicos/análise , Canais Iônicos/genética , Canais Iônicos/metabolismo , Cinesinas/análise , Cinesinas/metabolismo , Microscopia Eletrônica de Transmissão , Microtúbulos/metabolismo , Sondas Moleculares/química , Neurônios/metabolismo , Receptores de AMPA/análise , Receptores de AMPA/metabolismo , Anticorpos de Cadeia Única/químicaRESUMO
We developed a coating method to produce functionalized small quantum dots (sQDs), about 9â nm in diameter, that were stable for over a month. We made sQDs in four emission wavelengths, from 527 to 655â nm and with different functional groups. AMPA receptors on live neurons were labeled with sQDs and postsynaptic density proteins were visualized with super-resolution microscopy. Their diffusion behavior indicates that sQDs access the synaptic clefts significantly more often than commercial QDs.
Assuntos
Corantes Fluorescentes/análise , Neurônios/citologia , Pontos Quânticos/análise , Receptores de AMPA/análise , Animais , Células Cultivadas , Microscopia de Fluorescência , Imagem Óptica , RatosRESUMO
Aptamers, single-stranded nucleic acids that can selectively bind to various target molecules, have been widely used for constructing biosensors. A major challenge in this field, however, is direct sensing of analytes in complex biological media such as undiluted serum. While progress has been made in developing an inhomogeneous assay by using a preseparation step to wash away the interferences within serum, a facile strategy for direct detection of targets in homogeneous unprocessed serum is highly desired. We herein report a turn-on luminescent aptamer biosensor for the direct detection of adenosine in undiluted and unprocessed serum, by taking advantage of a terbium chelate complex with long luminescence lifetime to achieve time-resolved detection. The sensor exhibits a detection limit of 60 µM adenosine while marinating excellent selectivity that is comparable to those in buffer. The approach demonstrated here can be applied for direct detection and quantification of a broad range of analytes in biological media by using other aptamers.
Assuntos
Adenosina/sangue , Aptâmeros de Nucleotídeos/metabolismo , Complexos de Coordenação/química , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Térbio/química , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Quelantes/químicaRESUMO
Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).
Assuntos
Transferência de Energia , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Medições Luminescentes , Movimento , Oócitos/metabolismo , Potássio/metabolismo , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Superfamília Shaker de Canais de Potássio , Xenopus laevisRESUMO
Nicotinic acetylcholine receptors are some of the most studied synaptic proteins; however, many questions remain that can only be answered using single molecule approaches. Here we report our results from single α7 and neuromuscular junction type nicotinic acetylcholine receptors in mammalian cell membranes. By labeling the receptors with fluorophore-labeled bungarotoxin, we can image individual receptors and count the number of bungarotoxin-binding sites in receptors expressed in HEK 293 cells. Our results indicate that there are two bungarotoxin-binding sites in neuromuscular junction receptors, as expected, and five in α7 receptors, clarifying previous uncertainty. This demonstrates a valuable technique for counting subunits in membrane-bound proteins at the single molecule level, with nonspecialized optics and with higher signal/noise ratios than previous fluorescent protein-based techniques.
Assuntos
Bungarotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Sítios de Ligação , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Junção Neuromuscular/metabolismo , Fotodegradação , Receptor Nicotínico de Acetilcolina alfa7RESUMO
A quinoline sensitizer-centered lanthanide chelate system of novel design for TR-LRET was prepared; it exhibited high labelling efficiency with a his-tagged protein (ERalpha-LBD) on the Ni-NTA beads, using a mixed metal chelate protocol, and it functioned well in TR-LRET protein-protein interaction assays.
Assuntos
Quelantes/química , Transferência de Energia , Elementos da Série dos Lantanídeos/química , Ácido Nitrilotriacético/análogos & derivados , Compostos Organometálicos/química , Proteínas/química , Quinolinas/química , Coloração e Rotulagem/métodos , Transferência Ressonante de Energia de Fluorescência , Elementos da Série dos Lantanídeos/síntese química , Microesferas , Modelos Químicos , Ácido Nitrilotriacético/química , Quinolinas/síntese química , Fatores de TempoRESUMO
Polyaminocarboxylate-based luminescent lanthanide complexes have unusual emission properties, including millisecond excited-state lifetimes and sharply spiked spectra compared to common organic fluorophores. There are three distinct sections in the structure of the luminescent lanthanide chelates: a polyaminocarboxylate backbone to bind the lanthanide ions tightly, an antenna molecule to sensitize the emission of lanthanide ions, and a reactive group to attach to biomolecules. We have previously reported the modifications on the chelates, on the antenna molecules (commonly cs124), and on the reactive sites. In searching for stronger binding chelates and better protection from solvent hydration, here we report the modification of the coordination number of the chelates. A series of 9- and 10-dentate chelates were synthesized. Among them, the 1-oxa-4,7-diazacyclononane (N2O)-containing chelate provides the best protection to the lanthanide ions from solvent molecule attack, and forms the most stable lanthanide coordination compounds. The TTHA-based chelate provides moderately good protection to the lanthanide ions.
Assuntos
Quelantes/química , Éteres de Coroa/química , Ácido Edético/análogos & derivados , Elementos da Série dos Lantanídeos/química , Luminescência , Compostos Organometálicos/química , Quelantes/síntese química , Ácido Edético/química , Estrutura Molecular , Compostos Organometálicos/síntese química , Fatores de TempoRESUMO
Quantum dots are fluorescent nanoparticles with narrow-band, size-tunable, and long-lasting emission. Typical formulations used for imaging proteins in cells are hydrodynamically much larger than the protein targets, so it is critical to assess the impact of steric effects deriving from hydrodynamic size. This report analyzes a new class of quantum dots that have been engineered for minimized size specifically for imaging receptors in narrow synaptic junctions between neurons. We use fluorescence correlation spectroscopy and transmission electron microscopy to calculate the contributions of the crystalline core, organic coating, and targeting proteins (streptavidin) to the total hydrodynamic diameter of the probe, using a wide range of core materials with emission spanning 545-705 nm. We find the contributing thickness of standard commercial amphiphilic polymers to be ~8 to ~14 nm, whereas coatings based on the compact ligand HS-(CH2)11 - (OCH2CH2)4-OH contribute ~6 to ~9 nm, reducing the diameter by ~2 to ~5 nm, depending on core size. When the number of streptavidins for protein targeting is minimized, the total diameter can be further reduced by ~5 to ~11 nm, yielding a diameter of 13.8-18.4 nm. These findings explain why access to the narrow synapse derive primarily from the protein functionalization of commercial variants, rather than the organic coating layers. They also explain why those quantum dots with size around 14 nm with only a few streptavidins can access narrow cellular structures for neuronal labeling, whereas those >27 nm and a large number of streptavidins, cannot.
RESUMO
Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5-10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD 'slots', our findings suggest that AMPARs rapidly enter stable 'nanodomains' in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes.
Assuntos
Corantes Fluorescentes/metabolismo , Neurônios/metabolismo , Imagem Óptica/métodos , Densidade Pós-Sináptica/metabolismo , Receptores de AMPA/genética , Sinapses/metabolismo , Animais , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes/química , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Neurônios/ultraestrutura , Densidade Pós-Sináptica/ultraestrutura , Cultura Primária de Células , Transporte Proteico , Pontos Quânticos/química , Pontos Quânticos/metabolismo , Ratos , Receptores de AMPA/metabolismo , Coloração e Rotulagem/métodos , Sinapses/ultraestrutura , Fatores de TempoRESUMO
Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial enzyme which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG's to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20-30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes.
Assuntos
Técnicas Citológicas/métodos , Corantes Fluorescentes/metabolismo , Microscopia Intravital/métodos , Microscopia de Fluorescência/métodos , Proteínas/análise , Coloração e Rotulagem/métodos , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular , Sobrevivência Celular , Cricetinae , Glutationa/metabolismo , Humanos , Oxigenases/metabolismo , Estreptolisinas/metabolismoRESUMO
The synthesis, characterization, and reactivity of a new sulfur-rich tridentate ligand, tetrakis(2-thienyl)borate (1(-)()), are reported along with a molecular orbital analysis of its coordination to a metal center. Unlike the analogous tetrakis((methylthio)methyl)borate (2(-)()), 1(-)() does not coordinate Mo(CO)(3) when reacted with (C(7)H(8))Mo(CO)(3). The sulfur atoms in both ligands are oriented to coordinate the metal in a pyramidal eta(1) sulfur-bound mode. Approximate molecular orbital calculations are used to compare the metal-ligand interactions in these related species, and the results indicate that the magnitude and polarizability of the electronic charge density of the lone pairs on the sulfur atoms dictate the coordination strength of the ligands. Simple Mulliken atomic charges and orbital occupation numbers are used to determine the extent of charge delocalization. While the conjugation of the sulfur lone pair electrons with adjacent pi bonds in the ligands decreases the corresponding Lewis basicity, the contribution from the aromaticity in the thienyl groups is negligible. During the course of these studies, the structure of K[1] was determined by X-ray diffraction. K[1]: monoclinic space group C2/c, with a = 16.00(2) Å, b = 7.680(7) Å, c = 16.22(2) Å, beta = 118.520(7) degrees, V = 1750(3) Å(3), Z = 4, R(F) = 0.0494, and R(w)(F(2)()) = 0.122. The crystal lattice contains one-dimensional chains of 1(-)() bridged by K ions.
RESUMO
Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Células Procarióticas/química , Células Procarióticas/metabolismo , Canais de Sódio/química , Canais de Sódio/metabolismo , Proteínas de Bactérias/genética , Calibragem , Bicamadas Lipídicas , Modelos Moleculares , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Sódio/genéticaRESUMO
Luminescent lanthanide complexes have unusual spectroscopic characteristics, including millisecond excited-state lifetime and sharply spiked emission spectra. These characteristics make them valuable alternatives to conventional organic fluorescent probes in detection applications and for measuring nanometer-scale conformational changes in biomolecules via resonance energy transfer. Our group has previously reported the syntheses and application of various luminescence complexes that have polyaminocarboxylate chelates coupled to a carbostyril antenna and thiol or amine-reactive groups. Here we report the syntheses of new thiol-reactive forms of DTPA-cs124 chelates, including two iodoacetamide forms (phenylalanine-iodoacetamide and ethylenediamine iodoacetamide) and two methane-thio-sulfonate forms (ethylmethanethiosulfonate and carboxyethylmethanethiosulfonate). In addition, we have developed an improved synthesis of a previously reported maleimide form.