Inside-out regulation of E-cadherin conformation and adhesion.

Cadherin cell-cell adhesion proteins play key roles in tissue morphogenesis and wound healing. Cadherin ectodomains bind in two conformations, X-dimers and strand-swap dimers, with different adhesive properties. However, the mechanisms by which cells regulate ectodomain conformation are unknown.

Improving estimation of kinetic parameters in dynamic force spectroscopy using cluster analysis.

Dynamic Force Spectroscopy (DFS) is a widely used technique to characterize the dissociation kinetics and interaction energy landscape of receptor-ligand complexes with single-molecule resolution. In an Atomic Force Microscope (AFM)-based DFS experiment, receptor-ligand complexes, sandwiched between an AFM tip and substrate, are ruptured at different stress rates by varying the speed at which the AFM-tip and substrate are pulled away from each other. The rupture events are grouped according to their pulling speeds, and the mean force and loading rate of each group are calculated.

Minimizing open-loop piezoactuator nonlinearity artifacts in atomic force microscope measurements.

Atomic force microscopes (AFMs) are widely used to study molecular interactions with piconewton force sensitivity. In an AFM, interaction forces are measured by reflecting a laser beam off a cantilever onto a position sensitive detector and monitoring cantilever deflection. Precise measurements of interaction forces rely on accurately determining the optical lever sensitivity, i.

Copper-induced structural conversion templates prion protein oligomerization and neurotoxicity.

Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. Copper exposure has been linked to prion pathogenesis; however, its mechanistic basis is unknown. We resolve, with single-molecule precision, the molecular mechanism of Cu(2+)-induced misfolding of PrP under physiological conditions.

Fluorescence axial localization with nanometer accuracy and precision.

We describe a new technique, standing wave axial nanometry (SWAN), to image the axial location of a single nanoscale fluorescent object with sub-nanometer accuracy and 3.7 nm precision. A standing wave, generated by positioning an atomic force microscope tip over a focused laser beam, is used to excite fluorescence; axial position is determined from the phase of the emission intensity.

Zernike phase plate cryoelectron microscopy facilitates single particle analysis of unstained asymmetric protein complexes.

Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa).

Improvement in resolution of laser capture microdissection using near-field probe to capture nanoparticles.

Purpose: A modified laser capture microdissection (LCM) system is developed to improve resolution to 400 nm, using a laser light (808 nm) transmitted by a near-field tip probe.

Materials And Methods: Using a 150-nm aperture to heat an ethylene vinyl acetate (EVA) film, melted spots on the average of 400 nm in diameter are generated on the underlying target composed of a 20-nm gold-particle monolayer. The near-field tip probe composed of fiber is set on a 2-D nanometer piezoactuator (PZT) for precise capturing of the monolayer of gold particles.

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis.

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5' end of RNA transcript in the pol II elongation complex.