We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the α-hemolysin (α-HL) channel in its state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the protective antigen 63 (PA63) channel, locate where lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel.
View Article and Find Full Text PDFTethered lipid bilayer membranes (tBLMs) have been used in many applications, including biosensing and membrane protein structure studies. This report describes a biosensor for anthrax toxins that was fabricated through the self-assembly of a tBLM with B. anthracis protective antigen ion channels that are both the recognition element and electrochemical transducer.
View Article and Find Full Text PDFBinding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS.
View Article and Find Full Text PDFMeasurement of live-cell binding interactions is vital for understanding the biochemical reactions that drive cellular processes. Here, we develop, characterize, and apply a new procedure to extract information about binding to an immobile substrate from fluorescence correlation spectroscopy (FCS) autocorrelation data. We show that existing methods for analyzing such data by two-component diffusion fits can produce inaccurate estimates of diffusion constants and bound fractions, or even fail altogether to fit FCS binding data.
View Article and Find Full Text PDFWe report fluorescence correlation spectroscopy measurements of the translational diffusion coefficient of various probe particles in dilute and semidilute aqueous poly(vinyl alcohol) solutions. The range of sizes of the particles (fluorescent molecules, proteins, and polymers) was chosen to explore various length scales of the polymer solutions as defined by the polymer-polymer correlation length. For particles larger than the correlation length, we find that the diffusion coefficient, D, decreases exponentially with the polymer concentration.
View Article and Find Full Text PDFPhys Rev E Stat Nonlin Soft Matter Phys
January 2006
We have used small-angle light-scattering (SALS), microscopy, and measurements to study structural changes produced in unbuffered agarose gels as ions migrate under applied electric fields (3-20 V/cm). Anisotropic, bowtielike, light-scattering patterns were observed, whose development occurred more quickly at higher fields. The horizontal lobes were more pronounced at higher polymer concentration.
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