RESUMEN
It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through â¼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.
Asunto(s)
Nanoporos , Biosíntesis de Proteínas , ARN de Transferencia/química , Sitios de Unión , Electroforesis , Cinética , Aprendizaje Automático , ARN de Transferencia/genética , Ribosomas/química , Ribosomas/genéticaRESUMEN
We report the solution-phase synthesis and surface processing of ~2-5 µm long single-crystalline IV-VI tin(II) sulfide (SnS) ultrathin nanoribbons, with thicknesses down to 10 nm, and their use in single nanoribbon based photodetectors. The SnS nanoribbons grow via a metastable-to-stable phase transition from zinc blende (ZB) nanospheres to orthorhombic nanoribbons; dual-phase intermediate heterostructures with zinc blende nanosphere heads and orthorhombic nanoribbon tails were observed. Exchange of long, insulating organic oleylamine ligands by short, inorganic HS(-) ligands converts the organic SnS nanoribbons into completely inorganic, hydrophilic structures. Field-effect transistor (FET) devices were made from single SnS nanoribbons, both before and after ligand exchange, which exhibit p-type semiconductor behavior. The SnS single nanoribbon based photodetector devices showed highly sensitive and rapid photocurrent responses to illumination by blue, green, and red light. The switching behavior of photocurrent generation and annihilation is complete within approximately 1 ms and exhibits high photoconductivity gains (up to 2.3 × 10(4)) and good stability. The ON/OFF ratio of the photodetector can be engineered to 80 (4 nA/50 pA) using a small drain current (10 mV) for the all inorganic SnS nanoribbons. This work paves the way for the colloidal growth of low-cost, environmentally benign, single-crystalline narrow band gap semiconductor nanostructures from abundant elements for applications in photodetectors and other nanoscale devices.
RESUMEN
We report experimental results on the inactivation of encephalomyocarditis virus, M13 bacteriophage, and Salmonella typhimurium by a visible femtosecond laser. Our results suggest that inactivation of virus and bacterium by a visible femtosecond laser involves completely different mechanisms. Inactivation of viruses by a visible femtosecond laser involves the breaking of hydrogen∕hydrophobic bonds or the separation of the weak protein links in the protein shell of a viral particle. In contrast, inactivation of bacteria is related to the damage of their DNAs due to irradiation of a visible femtosecond laser. Possible mechanisms for the inactivation of viruses and bacteria are discussed.
Asunto(s)
Bacteriófago M13/efectos de la radiación , Virus de la Encefalomiocarditis/efectos de la radiación , Láseres de Estado Sólido/uso terapéutico , Salmonella typhimurium/efectos de la radiación , Animales , Bovinos , Dicroismo Circular , ADN Viral/efectos de la radiación , Microscopía de Fuerza Atómica , Microscopía de Fluorescencia por Excitación Multifotónica , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de la radiación , Espectrometría de Fluorescencia , Espectrometría Raman , Virión/efectos de la radiaciónRESUMEN
Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Pollos , ADN/metabolismo , Células HeLa , Humanos , Microscopía de Fuerza Atómica , Proteínas Mutantes/metabolismo , Nucleosomas/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Electricidad Estática , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
Eukaryotic DNA is packaged into chromatin, which regulates genome activities such as telomere maintenance. This study focuses on the interactions of a myb/SANT DNA-binding domain from the telomere-binding protein, TRF2, with reconstituted telomeric nucleosomal array fibers. Biophysical characteristics of the factor-bound nucleosomal arrays were determined by analytical agarose gel electrophoresis (AAGE) and single molecules were visualized by atomic force microscopy (AFM). The TRF2 DNA-binding domain (TRF2 DBD) neutralized more negative charge on the surface of nucleosomal arrays than histone-free DNA. Binding of TRF2 DBD at lower concentrations increased the radius and conformational flexibility, suggesting a distortion of the fiber structure. Additional loading of TRF2 DBD onto the nucleosomal arrays reduced the flexibility and strongly blocked access of micrococcal nuclease as contour lengths shortened, consistent with formation of a unique, more compact higher-order structure. Mirroring the structural results, TRF2 DBD stimulated a strand invasion-like reaction, associated with telomeric t-loops, at lower concentrations while inhibiting the reaction at higher concentrations. Full-length TRF2 was even more effective at stimulating this reaction. The TRF2 DBD had less effect on histone-free DNA structure and did not stimulate the t-loop reaction with this substrate, highlighting the influence of chromatin structure on the activities of DNA-binding proteins.
Asunto(s)
Nucleosomas/química , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , ADN/metabolismo , Humanos , Nucleasa Microcócica , Microscopía de Fuerza Atómica , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Oligonucleótidos/análisis , Estructura Terciaria de Proteína , Proteína 2 de Unión a Repeticiones Teloméricas/químicaRESUMEN
We report the in vitro selection of DNA aptamers that bind to histone H4 proteins acetylated at lysine 16. The best aptamer identified in this selection binds to the target protein with a K(d) of 21 nM and discriminates against both the nonacetylated protein and histone H4 proteins acetylated at lysine 8. Comparative binding assays performed with a chip-quality antibody reveal that this aptamer binds to the acetylated histone target with similar affinity to a commercial antibody but shows significantly greater specificity (15-fold versus 2400-fold) for the target molecule. This result demonstrates that aptamers that are both modification and location specific can be generated to bind specific protein post-translational modifications.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Histonas/metabolismo , Acetilación , Sitios de Unión , Humanos , Lisina , Unión Proteica , Procesamiento Proteico-Postraduccional , Especificidad por SustratoRESUMEN
We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. The laser technology targets the global mechanical properties of the viral protein shell, making it relatively insensitive to the local genetic mutation in the target viruses. As a result, the approach can inactivate both the wild and mutated strains of viruses. This intriguing advantage is particularly important in the treatment of diseases involving rapidly mutating viral species such as HIV. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.
Asunto(s)
Rayos Láser , Óptica y Fotónica/métodos , Espectroscopía Infrarroja Corta/métodos , Inactivación de Virus/efectos de la radiación , Virus/efectos de la radiación , Alphapapillomavirus/fisiología , Alphapapillomavirus/efectos de la radiación , Animales , Bacteriófago M13/fisiología , Bacteriófago M13/efectos de la radiación , Células Cultivadas , Células Dendríticas/efectos de la radiación , Eritrocitos/efectos de la radiación , VIH/fisiología , VIH/efectos de la radiación , Humanos , Células Jurkat/efectos de la radiación , Ratones , Microscopía de Fuerza Atómica , Virus del Mosaico del Tabaco/fisiología , Virus del Mosaico del Tabaco/efectos de la radiaciónRESUMEN
Atomic force microscopy (AFM) can directly visualize single molecules in solution, which makes it an extremely powerful technique for carrying out studies of biological complexes and the processes in which they are involved. A recent development, called Recognition Imaging, allows the identification of a specific type of protein in solution AFM images, a capability that greatly enhances the power of the AFM approach for studies of complex biological materials. In this technique, an antibody against the protein of interest is attached to an AFM tip. Scanning a sample with this tip generates a typical topographic image simultaneously and in exact spatial registration with a "recognition image." The latter identifies the locations of antibody-antigen binding events and thus the locations of the protein of interest in the image field. The recognition image can be electronically superimposed on the topographic image, providing a very accurate map of specific protein locations in the topographic image. This technique has been mainly used in in vitro studies of biological complexes and reconstituted chromatin, but has great potential for studying chromatin and protein complexes isolated from nuclei.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Cromatina/metabolismo , Microscopía de Fuerza Atómica/métodos , Animales , Humanos , Nucleosomas/metabolismoRESUMEN
The glycosylation state of individual antibodies was imaged using an atomic force microscope with a probe modified with lectins and an image acquisition system that permits simultaneous acquisition of sample topography data along with a map of lectin binding sites.
Asunto(s)
Anticuerpos/química , Glicosilación , Microscopía de Fuerza Atómica/métodos , Sitios de Unión , Humanos , Lectinas , Sondas MolecularesRESUMEN
Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Fragmentos de Inmunoglobulinas/química , Insulina/inmunología , Microscopía de Fuerza Atómica/métodos , Ensayo de Inmunoadsorción Enzimática , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/inmunología , Mutagénesis Sitio-DirigidaRESUMEN
Recognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. The technique relies on the use of antibodies tethered to the cantilever tip of an AFM probe to detect cognate antigens deposited onto a mica surface. Despite the power of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of suitable quality antibodies. Here we report the in vitro selection and recognition imaging of anti-histone H4 aptamers. In addition to identifying aptamers to highly basic proteins, these results suggest that aptamers provide an efficient, cost-effective route to highly selective affinity reagents for recognition imaging microscopy.
Asunto(s)
Histonas/química , Secuencia de Aminoácidos , Histonas/ultraestructura , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Telomeres are DNA-protein complexes at the ends of eukaryotic chromosomes, the integrity of which is essential for chromosome stability. An important telomere binding protein, TTAGGG repeat factor 2 (TRF2), is thought to protect telomere ends by remodeling them into T-loops. We show that TRF2 specifically interacts with telomeric ss/ds DNA junctions and binding is sensitive to the sequence of the 3', guanine-strand (G-strand) overhang and double-stranded DNA sequence at the junction. Association of TRF2 with DNA junctions hinders cleavage by exonuclease T. TRF2 interactions with the G-strand overhang do not involve the TRF2 DNA binding domain or the linker region. However, mobility shifts and atomic force microscopy show that the previously uncharacterized linker region is involved in DNA-specific, TRF2 oligomerization. We suggest that T-loop formation at telomere ends involves TRF2 binding to the G-strand overhang and oligomerization through both the known TRFH domain and the linker region.
Asunto(s)
Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/química , Secuencia de Bases , Sitios de Unión , Datos de Secuencia Molecular , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen.
Asunto(s)
Anticuerpos Monoclonales/química , Codón , Genes Supresores , Fragmentos de Péptidos/química , Biblioteca de Péptidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Reacciones Antígeno-Anticuerpo , Histonas/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estreptavidina/químicaRESUMEN
Carotenoids (Car) act as "wires" that discharge unwanted electrons in the reaction center of higher plants. One step in this "side-path" electron conduction is thought to be mediated by Car oxidation. We have carried out direct measurements of the conductance of single-Car molecules under potential control in a membrane-mimicking environment, and we found that when Car are oxidized conductance is enhanced and the electronic decay constant (beta) is decreased. However, the neutral molecule may already be conductive enough to account for observed electron transfer rates.
Asunto(s)
Carotenoides/metabolismo , Conductividad Eléctrica , Carotenoides/química , Transporte de Electrón , ElectronesRESUMEN
The conductance of single alkanedithiols covalently bound to gold electrodes has been studied by statistical analysis of repeatedly created molecular junctions. For each molecule, the conductance histogram reveals two sets of well-defined peaks, corresponding to two different conductance values. We have found that (1) both conductance values decrease exponentially with the molecular length with an identical decay constant, beta approximately equal to 0.84 A(-1), but with a factor of 5 difference in the prefactor of the exponential function. (2) The current-voltage curves of the two sets can be fit with the Simmons tunneling model. (3) Both conductance values are independent of temperature (between -5 and 60 degrees C) and the solvent. (4) Despite the difference in the conductance, the forces required to break the molecular junctions are the same, 1.5 nN. These observations lead us to believe that the conduction mechanism in alkanedithiols is due to electron tunneling or superexchange via the bonds along the molecules, and the two sets of conductance peaks are due to two different microscopic configurations of the molecule-electrode contacts.
RESUMEN
Negative differential resistance (NDR) peaks in the current-voltage characteristics of ferrocenylundecanethiol self-assembled monolayers are not reversible. The peaks turn to smoothly increasing currents as oxygen is removed from the system, indicating that NDR arises from the reaction of an energetic charged species with ambient oxygen.
Asunto(s)
Avidina/química , Biotina/química , Microscopía de Fuerza Atómica/métodos , Microscopía/métodos , Tomografía/métodos , Animales , Biofisica/métodos , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Oscilometría , Programas Informáticos , Estrés Mecánico , Tomografía/instrumentaciónRESUMEN
The conductance of carotenoid polyenes chemically bound at each end to gold contacts has been measured for single molecules containing 5, 7, 9, and 11 carbon-carbon double bonds in conjugation. The electronic decay constant, beta, is determined to be 0.22 +/- 0.04 A-1, in close agreement with the value obtained from first principles simulations (0.22 +/- 0.01 A-1). The absolute values of the molecular conductance are within a factor of 3 of those calculated from first principles. The small value of beta demonstrates that conductivity drops off only slowly with chain length, confirming that carotenoid conjugated chains are relatively good molecular "wires".
RESUMEN
Stochastic on-off conductivity switching observed in phenylene-ethynylene oligomers has been explained in terms of changes in ring conformations, or electron localization, or both. We report the observation of stochastic on-off switching in the simplest of wired molecules: octanedithiol, decanedithiol, and dodecanedithiol bonded on an Au(111) surface. Stochastic switching was observed even when a top gold contact was pressed on by a conducting atomic force microscope tip at constant force. The rate of switching increased substantially at 60 degrees C, a temperature at which these films are commonly annealed. Because such switching in alkanethiols is unlikely to be caused by internal molecular electronic changes and cannot be fully accounted for by breaking of the top contact, we argue that the cause is the well-known mobility of molecules tethered to gold via a thiol linkage.
RESUMEN
We have found that mica surfaces functionalized with aminopropyltriethoxysilane and aldehydes bind chromatin strongly enough to permit stable and reliable solution imaging by atomic force microscopy. The method is highly reproducible, uses very small amounts of material, and is successful even with very light degrees of surface modification. This surface is far superior to the widely used aminopropyltriethoxysilane-derivatized mica surface and permits resolution of structure on the nanometer-scale in an aqueous environment, conditions that are particularly important for chromatin studies. For example, bound nucleosomal arrays demonstrate major structural changes in response to changes in solution conditions, despite their prior fixation (to maintain nucleosome loading) and tethering to the surface with glutaraldehyde. By following individual molecules through a salt titration in a flow-through cell, one can observe significant changes in apparent nucleosome size at lower [salt] and complete loss of DNA from the polynucleosomal array at high salt. The latter result demonstrates that the DNA component in these arrays is not constrained by the tethering. The former result is consistent with the salt-induced loss of histones observed in bulk solution studies of chromatin and demonstrates that even histone components of the nucleosome are somewhat labile in these fixed and tethered arrays. We foresee many important applications for this surface in future atomic force microscopy studies of chromatin.