RESUMO
Pulmonary surfactant protein D (SP-D) is a glycoprotein from the collectin family that is a component of the lung surfactant system. It exhibits host defense and immune regulatory functions in addition to contributing to the homeostasis of the surfactant pool within the alveolar airspaces. It is known that the SP-D monomer forms trimers, which further associate into higher-order oligomers. However, the pathway and the interactions involved in the assembly of SP-D oligomers are not clearly understood. In the current study, a recombinant form of full-length human SP-D (rhSP-D) has been qualitatively and quantitatively studied by atomic force microscopy (AFM) and electrophoresis, with the aim to understand the conformational diversity and the determinants defining the oligomerization of the protein. The rhSP-D preparation studied is a mixture of trimers, hexamers, dodecamers and higher-order oligomeric species, with dodecamers accounting for more than 50% of the protein by mass. Similar structures were also found in hSP-D obtained from proteinosis patients, with the largest fuzzy-ball-like oligomers being more abundant in these samples. The proportion of dodecamer is increased under acidic conditions, accompanied by a conformational change into more compact configurations. Two hexamers appear to be the minimal necessary unit for dodecamer formation, with stabilization of the dodecamer occurring via non-covalent, ionic, and hydrophobic interactions between the individual N-terminal domains and the proximal area of the SP-D collagen stems.
Assuntos
Proteinose Alveolar Pulmonar/metabolismo , Proteína D Associada a Surfactante Pulmonar/química , Proteína D Associada a Surfactante Pulmonar/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Microscopia de Força Atômica , Modelos Moleculares , Multimerização Proteica , TermodinâmicaRESUMO
Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.
RESUMO
Atomic force microscopy can potentially provide information on protein volumes, shapes, and interactions but is susceptible to variable tip-induced artifacts. In this study, we present an atomic force microscopy approach that can measure volumes of nonglobular polypeptides such as structural maintenance of chromosomes (SMC) proteins, and use it to study the interactions that occur within and between SMC complexes. Together with the protein of interest, we coadsorb a DNA molecule and use it as a fiducial marker to account for tip-induced artifacts that affect both protein and DNA, allowing normalization of protein volumes from images taken on different days and with different tips. This approach significantly reduced the error associated with volume analysis, and allowed determination of the oligomeric states and architecture of the Bacillus subtilis SMC complex, formed by the SMC protein, and by the smaller ScpA and ScpB subunits. This work reveals that SMC and ScpB are dimers and that ScpA is a stable monomer. Moreover, whereas ScpA binds directly to SMC, ScpB only binds to SMC in the presence of ScpA. Notably, the presence of both ScpA and ScpB favored the formation of higher-order structures of SMC complexes, suggesting a role for these subunits in the organization of SMC oligomers.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA/química , Microscopia de Força Atômica , Sondas Moleculares/química , Adsorção , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peso Molecular , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
Over the past few years, it has become increasingly apparent that double-stranded RNA (dsRNA) plays a far greater role in the life cycle of a cell than previously expected. Numerous proteins, including helicases, polymerases, and nucleases interact specifically with the double helix of dsRNA. To understand the detailed nature of these dsRNA-protein interactions, the (bio)chemical, electrostatic, and mechanical properties of dsRNA need to be fully characterized. We present measurements of the persistence length of dsRNA using two different single-molecule techniques: magnetic tweezers and atomic force microscopy. We deduce a mean persistence length for long dsRNA molecules of 63.8 +/- 0.7 nm from force-extension measurements with the magnetic tweezers. We present atomic force microscopy images of dsRNA and demonstrate a new method for analyzing these, which yields an independent, yet consistent value of 62 +/- 2 nm for the persistence length. The introduction of these single-molecule techniques for dsRNA analysis opens the way for real-time, quantitative analysis of dsRNA-protein interactions.
Assuntos
Biofísica/métodos , Microscopia de Força Atômica/métodos , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/ultraestrutura , Biofísica/instrumentação , Soluções Tampão , DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Eletroforese em Gel de Ágar , Cinética , Magnetismo , Microscopia de Força Atômica/instrumentação , Modelos Biológicos , Modelos Estatísticos , Modelos Teóricos , Reação em Cadeia da Polimerase , Ligação Proteica , RNA/química , Ribonuclease III/química , Eletricidade Estática , Temperatura , Transcrição Gênica , Proteínas Virais/metabolismoRESUMO
OBJECTIVE: To determine the features of hearing loss due to the Q829X mutation in the OTOF gene, the third most frequent mutation causing prelingual deafness reported so far in the Spanish population. MATERIALS AND METHODS: We carried out genetic characterisation of 16 individuals from a consanguineous family from Cantabria, in which 4 members were affected by deafness. RESULTS: All 4 hearing impaired individuals were homozygous for the Q829X mutation in the OTOF gene. The auditory defect was a profound, bilateral, symmetrical, sensorineural hearing loss of prelingual onset. No other clinical alterations were observed. Individuals heterozygous for the Q829X mutation were unaffected. CONCLUSIONS: The Q829X mutation in the OTOF gene causes severe to profound sensorineural hearing loss of prelingual onset. Early detection of individuals carrying this mutation is important for the application of palliative treatment and special education.
Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
The capabilities of the atomic force microscope for imaging biomolecules under physiological conditions has been systematically investigated. Contact, dynamic, and jumping modes have been applied to four different biological systems: DNA, purple membrane, Alzheimer paired helical filaments, and the bacteriophage phi29. These samples have been selected to cover a wide variety of biological systems in terms of sizes and substrate contact area, which make them very appropriate for the type of comparative studies carried out in the present work. Although dynamic mode atomic force microscopy is clearly the best choice for imaging soft samples in air, in liquids there is not a leading technique. In liquids, the most appropriate imaging mode depends on the sample characteristics and preparation methods. Contact or dynamic modes are the best choices for imaging molecular assemblies arranged as crystals such as the purple membrane. In this case, the advantage of image acquisition speed predominates over the disadvantage of high lateral or normal force. For imaging individual macromolecules, which are weakly bonded to the substrate, lateral and normal forces are the relevant factors, and hence the jumping mode, an imaging mode which minimizes lateral and normal forces, is preferable to other imaging modes.
Assuntos
Fagos Bacilares/ultraestrutura , DNA/ultraestrutura , Aumento da Imagem/métodos , Microscopia de Força Atômica/métodos , Emaranhados Neurofibrilares/ultraestrutura , Membrana Purpúrea/ultraestrutura , SoluçõesRESUMO
Paired helical filaments (PHF) is an aberrant structure present in the brain of Alzheimer's disease patients which has been correlated with their degree of dementia. In order to determine the structure of PHF, several studies have been performed using atomic force microscopy (AFM). However, those studies have the limitation that they have not been done in solution and the sample could be far from the real physiological conditions. In this work we present an AFM analysis of PHF in liquid environment and we compare that analysis with that performed in dry conditions. PHF imaging in liquid was only possible by using jumping mode AFM as the imaging technique. Jumping mode AFM images of PHF in solution show first, a notable increase in the absolute values of the height of the filament, and second, a smaller ratio between the height measured at the upper and at the lower part of the PHF. Direct comparison of the experimental data with structural models has been performed. From this we conclude that the PHF structure is compatible with two coupled ribbons with an overall height of 20 nm and a width of 10 nm.
Assuntos
Microscopia de Força Atômica/métodos , Modelos Moleculares , Emaranhados Neurofibrilares/química , Emaranhados Neurofibrilares/ultraestrutura , Proteínas tau/química , Proteínas tau/ultraestrutura , Líquidos Corporais/química , Simulação por Computador , Conformação Proteica , Estrutura Secundária de Proteína , SoluçõesRESUMO
The measured height of DNA molecules adsorbed on a mica substrate by scanning probe microscopy is always less than the theoretical diameter. In this paper we show that, when imaged in ambient conditions, the molecules are usually immersed in the salt layer used to adsorb them to the substrate. This layer distorts the measurement of DNA height and is the main source of error but not the only one. We have performed different experiments to study this problem using two scanning force techniques: non-contact tapping mode in air and jumping mode in aqueous solution, where the dehydration phenomena is minimized. Height measurements of DNA in air using tapping mode reveal a height of 0.7+/-0.2nm. This value increases up to 1.5+/-0.2nm when the salt layer, in which the molecules are embedded, is removed. Jumping experiments in water give a value of 1.4+/-0.3nm when the maximum applied force is 300pN and 1.8+/-0.2nm at very low forces, which confirms the removal of the salt layer. Still, in all our experiments, the measured height of the DNA is less than the theoretical value. Our results show that although the salt layer present is important, some sample deformation due to either the loading force of the tip or the interaction with the substrate is also present.
Assuntos
DNA/ultraestrutura , Microscopia de Força Atômica/métodos , Adsorção , Silicatos de Alumínio , Umidade , SoluçõesRESUMO
A fundamental requirement for a molecule to be considered a molecular wire (MW) is the ability to transport electrical charge with a reasonably low resistance. We have carried out two experiments that measure first, the charge transfer from an electrode to the molecule, and second, the dielectric response of the MW. The latter experiment requires no contacts to either end of the molecule. From our experiments we conclude that adsorbed individual DNA molecules have a resistivity similar to mica, glass, and silicon oxide substrates. Therefore adsorbed DNA is not a conductor, and it should not be considered as a viable candidate for MW applications. Parallel studies on other nanowires, including single-walled carbon nanotubes, showed conductivity as expected.
Assuntos
DNA/química , DNA/ultraestrutura , Condutividade Elétrica , Microscopia de Força AtômicaRESUMO
In the presence of glucose the protein hexokinase 2 (Hxk2p), normally resident in the cytosol, is translocated to the nucleus where it impairs the activation of transcription of the glucose-repressed genes HXK1, GLK1 and SUC2, and promotes the activation of transcription of the glucose-induced genes HXK2 and HXT1. Here, we demonstrate the involvement of an heptameric motif, named the MED8 site, in the direct binding of the mediator protein Med8p, either as a monomer or as a homodimer. Because this site was previously involved in the Hxk2p-dependent glucose-induced regulation of gene transcription, we tested whether Hxk2p interacts with Med8p. Our results show that Hxk2 and Med8 proteins are physically associated and that this Hxk2p-Med8p interaction is of physiological significance because both proteins have been found interacting together in a cluster with DNA fragments containing the MED8 site. We conclude that Hxk2p operates through the MED8 site, by interacting with Med8p, in the glucose signal transduction pathway of Saccharomyces cerevisiae.
Assuntos
Glucose/farmacologia , Hexoquinase/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Genes Fúngicos/genética , Hexoquinase/química , Hexoquinase/genética , Complexo Mediador , Microscopia de Força Atômica , Testes de Precipitina , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
OBJECTIVE: To examine the audiometric patterns of familial hearing impairment due to the A1555G mutation in the mitochondrial DNA. PATIENTS AND METHODS: We include 55 subjects with the A1555G mutation from 6 unrelated families, affected by nonsyndromic sensorineural hearing loss and residing in Cantabria. The A1555G mutation was found in homoplasmy in all the families, except in one family, in which it was in heteroplasmy. Aside from standard history taking and general otolaryngological examination, pure tone audiometry was carried out in all patients. RESULTS: Hearing loss was developed by most of the patients. The auditory defect was a slowly progressive bilateral symmetrical sensorineural hearing loss, affecting mainly the high frequencies. In patients in which aminoglycoside ototoxicity could be excluded, hearing loss usually ranged from mild to moderate, with a late onset. In 17 cases there were previous history of treatment with a ototoxic drugs, and most of them developed severe hearing loss. One of them was deaf-mute. No audometric differences between families with the homoplasmic and the heteroplasmic A1555G mutation were observed. CONCLUSIONS: Patients with the A1555G mutation generally present bilateral symmetrical sensorineural hearing loss, ranging from mild to moderate, slowly progressive, which is obvious approximately in the second decade of life and affects specially the high frequencies. Hearing loss severity is increased by treatment with aminoglycosides.
Assuntos
Audiometria , DNA Mitocondrial/genética , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/fisiopatologia , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , LinhagemRESUMO
Regulation of gene expression is fundamental in biological systems. A systematic search for protein binding sites in gene promoters has been done in recent years. Biochemical techniques are easy and reliable when analysing protein interactions with short pieces of DNA, but are difficult and tedious when long pieces of DNA have to be analysed. Here we propose AFM as a reliable and easy technique for identifying protein interaction sites in long DNA molecules like gene promoters. We support this idea using a well-known model: the interaction of the Pho4 protein with the PHO5 gene promoter. We have also applied the technique to demonstrate that Mig1 protein binds to two motifs in the promoter of HXK2 gene. Our results allow us to define Mig1p as a new factor probably contributing to the carbon source-dependent transcription regulation of HXK2 gene.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Fosfato , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Escherichia coli , Microscopia de Força Atômica/métodos , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/ultraestrutura , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The electrical conductivity of biomaterials on a molecular scale is of fundamental interest in the life sciences. We perform first principles electronic structure calculations, which clearly indicate that lambda-DNA chains should present large resistance values. We also present two direct procedures to measure electrical currents through DNA molecules adsorbed on mica. The lower limit for the resistivity is 10(6) Omega . cm, in agreement with our calculations. We also show that low energy electron bombardment induces a rapid contamination and dramatically affects the measured conductivity, thus providing an explanation to recent reports of high DNA conductivity.
Assuntos
DNA/química , Animais , Humanos , Eletricidade EstáticaRESUMO
Med8 protein is a regulator that specifically binds to upstream activating sequences (UASs) of SUC2 promoter, to downstream repressing sequences (DRSs) of the HXK2 gene and to the carboxy-terminal domain of the RNA polymerase II. Atomic force microscopy has allowed for direct visualization of Med8 interactions with a 305 bp fragment of SUC2 promoter and with a 676 bp fragment of HXK2 gene, containing respectively the UASs and DRSs regulatory regions. This approach has provided complementary information about the position and the structure of the DNA-protein complexes. Med8 binding to DNA results in total covering of one of the two existing 7 bp motives (consensus, (A/C)(A/G)GAAAT) in the studied DNA fragments. No preference for binding either of the two UASs of SUC2 promoter as well as for the two DRSs of HXK2 gene has been found. We also discuss whether this protein works as dimer or as a monomer.