RESUMEN
We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of lambda DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the long-scale structure of the bacterial nucleoid.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , ADN/química , Proteínas Bacterianas/genética , Bacteriófago lambda/genética , ADN Viral/química , Proteínas de Unión al ADN/genética , Elasticidad , Factores de Integración del Huésped , Mutagénesis Sitio-DirigidaRESUMEN
We study experimentally a coiling instability of cylindrical multilamellar stacks of phospholipid membranes, induced by polymers with hydrophobic anchors grafted along their hydrophilic backbone. Our system is unique in that coils form in the absence of both twist and adhesion. We interpret our experimental results in terms of a model in which local membrane curvature and polymer concentration are coupled. The model predicts the occurrence of maximally tight coils above a threshold polymer occupancy. A proper comparison between the model and experiment involved imaging of projections from simulated coiled tubes with maximal curvature and complicated torsions.
RESUMEN
We have studied the pearling instability induced on hollow tubular lipid vesicles by hydrophilic polymers with hydrophobic side groups along the backbone. The results show that the polymer concentration is coupled to local membrane curvature. The relaxation of a pearled tube is characterized by two different well-separated time scales, indicating two physical mechanisms. We present a model, which explains the observed phenomena and predicts polymer segregation according to local membrane curvature at late stages.
Asunto(s)
Liposomas/química , Membranas Artificiales , Modelos Biológicos , Fosfatidilcolinas/química , Fosfolípidos/química , Colorantes Fluorescentes , Polímeros/químicaRESUMEN
We introduce a method for detecting and tracking small particles in a solution near a surface. The method is based on blocking the backreflected illumination beam in an objective-type total internal reflection microscope, leaving unhindered the light scattered by the particles and resulting in dark-field illumination. Using this method, we tracked the motion of 60-nm polystyrene beads with a signal-to-noise ratio of 6 and detected 20-nm gold particles with a signal-to-noise ratio of 5. We illustrate the method's use by following the Brownian motion of small beads attached by short DNA tethers to a substrate.
RESUMEN
We present a new approach to probing single-particle dynamics that uses dynamic light scattering from a localized region. By scattering a focused laser beam from a micron-size particle, we measure its spatial fluctuations via the temporal autocorrelation of the scattered intensity. We demonstrate the applicability of this approach by measuring the three-dimensional force constants of a single bead and a pair of beads trapped by laser tweezers. The scattering equations that relate the scattered intensity autocorrelation to the particle position correlation function are derived. This technique has potential applications for measurement of biomolecular force constants and probing viscoelastic properties of complex media.