We present unzipping force analysis of protein association (UFAPA) as a novel and versatile method for detection of the position and dynamic nature of protein-DNA interactions. A single DNA double helix was unzipped in the presence of DNA-binding proteins using a feedback-enhanced optical trap. When the unzipping fork in a DNA reached a bound protein molecule we observed a dramatic increase in the tension in the DNA, followed by a sudden tension reduction. Analysis of the unzipping force throughout an unbinding "event" revealed information about the spatial location and dynamic nature of the protein-DNA complex. The capacity of UFAPA to spatially locate protein-DNA interactions is demonstrated by noncatalytic restriction mapping on a 4-kb DNA with three restriction enzymes (BsoBI, XhoI, and EcoRI). A restriction map for a given restriction enzyme was generated with an accuracy of approximately 25 bp. UFAPA also allows direct determination of the site-specific equilibrium association constant (K(A)) for a DNA-binding protein. This capability is demonstrated by measuring the cation concentration dependence of K(A) for EcoRI binding. The measured values are in good agreement with previous measurements of K(A) over an intermediate range of cation concentration. These results demonstrate the potential utility of UFAPA for future studies of site-specific protein-DNA interactions.

Thermodynamic relaxation can generate complex nanostructured materials via self-assembly; these structures, however, are ultimately limited by chemical equilibria and diffusional transport processes. [1] In contrast, living systems use a concerted combination of thermodynamic and energydissipating processes to remove these functional limitations, and generate complex, structured materials with a wide range of adaptive and emergent behaviors. An underlying principle of such systems involves the dynamic self-assembly of materials, which occurs outside of thermodynamic equilibrium, requires a source of energy, and bridges multiple length scales. [2][3][4] Analogous principles have been applied to assemble a broad range of artificial ''dissipative'' structures [5] through electrorheological, [6] magnetohydrodynamic, [7] electrohydrodynamic, [8] and magnetorheological, [9] interactions that induce spatiotemporal ordering. Efforts to understand these effects have led to significant insights into fundamental nonequilibrium physics.[10] While these approaches expand the practical range of materials, they rely on programmed or user-defined stimuli to drive the assemblies out of equilibrium. The next major step in developing materials assemblies will involve selfregulating systems that define the dynamic assembly and adaptive behavior of materials. Such feedback-regulated systems will extend the functional nature of nanostructured materials to include revolutionary behaviors (e.g., adaptive reconfiguration and self-healing), currently unattainable by conventional self-assembly methods.There are few examples of dynamic self-assembly in which the energy component is intrinsic to the system, as opposed to externally applied (e.g., electromagnetic fields). One system involves the dynamic assembly of nanospools [11,12] and nanocomposite rings [13] wherein assembly is achieved through a stochastic interaction of energy-dissipation and thermodynamic processes. One remarkable characteristic of both structures is the significant energy (i.e., >10 5 kT) that is required for their formation, which is based on the relatively high bending rigidity of the microtubules. [11] This energy input is cooperatively supplied through the hydrolysis of ATP by kinesin (energy dissipation) and the formation of multiple biotin-streptavidin bonds (thermodynamic). In addition, the nanospools display a highly nonequilibrium existence in which the unzipping of biotin-streptavidin bonds by kinesin motor leads to the spontaneous unspooling for the structures.[11] A unique aspect of the nanocomposite rings concerns the ability to assemble quantum dots across multiple length scales. The microtubules in these structures serve as a nanoscale scaffold for assembling the quantum dots; the quantum dot-laden microtubules subsequently self-organize into microscale, optically active structures. [13] While the properties of these nonequilibrium structures have been described, the mechanism of their formation is unknown and may provide valuable insight with respect t...

We demonstrate the first site-specific single-molecule characterization of the prominent activation barrier for the disruption of a protein-DNA binding complex. We achieved this new capability by combining dynamic force spectroscopy with unzipping force analysis of protein association and used the combination to investigate restriction enzyme binding to specific DNA sites. Analysis revealed lifetimes and interaction distances for three protein-DNA interactions. This new method is able to distinguish protein-DNA binding complexes on a site-specific, single-molecule basis.

In this study, we report differences in the observed gliding speed of microtubules dependent on the choice of bovine casein used as a surface passivator. We observed differences in both speed and support of microtubules in each of the assays. Whole casein, comprised of αs1, αs2, β, and κ casein, supported motility and averaged speeds of 966±7 nm/s. Alpha casein can be purchased as a combination of αs1 and αs2 and supported gliding motility and average speeds of 949±4 nm/s. Beta casein did not support motility very well and averaged speeds of 870±30 nm/s. Kappa casein supported motility very poorly and we were unable to obtain an average speed. Finally, we observed that mixing alpha, beta, and kappa casein with the proportions found in bovine whole casein supported motility and averaged speeds of 966±6 nm/s.