Thirty-seventh Annual Meeting February 14-18, 1993 Washington Convention Center Washington, D.C. Wednesday Symposia and Posters, Part III
The interaction of the kInesin motor domain with the microtubule surface lattice was examined by electron microscopy of negatively stained and frozen-hydrated specimens. The N-terminal 401 amino adds of the DrosoEhila kinesin heavy chain (K401) which contains both the ATP and microtubule binding domains, were expressed in E. cli and purified to homogeneity as soluble, fully active, protein. This tnmncated monomeric form of kInesin made it possible to decorate individual microtubules heavily
... out cross-linking them into bundles. Complexes were formwd by mixing taxol-stabilized microtubules and K401 at I to 3 fines the mdar concentration of tubulin, pelleted, and analyzed by gel electrophoresis. Saturating binding was found to conrespond to one molecule of K401 per tubulin diner. According to both conventional negative staning and cryo-electron microscopy, the complexes were coated with regular patterns of bound K401 molecules with an axial repeat of 8nm. Optical diffraction of decorated microtubules showed a strong layerline at this spacing, confirming that one kinesi head bids per tubulin heterdimer. The addition of ATP to the K401-microtubule complex led to complete dissociation of kinesin from the microtubule surface. Mucociliary clearance is the primary mechanism by which inhaled foreign particles, including bacteria, are removed from the airways and lung. This clearance depends upon both the mucus secreted by the goblet cells and the microtubule-based ciliary beat frequency (CBF) of airway epithelial cells. To determine changes in CBF and [Ca2+]i of the same cell, tracheal epithelial cells were obtained from sheep by dissociation with protease and grown in culture for 2-14 days. Cells were imaged with a 10OX Fluor DL oil objective, enabling single cilia to be clearly observed. CBF was measured by online FF1 analysis of intensity changes of single pixels from digitized phase contrast microscopy images. Using a video camera with RS-170 timing, frequency response is limited to <15Hz since each pixel represents a 1/30s sampling interval. With 128 samples per FFT (=4.26s), the magnitude spectra usually showed a clear single peak. At 20'C, this peak frequency was between 5Hz and 10 Hz and was stable (±lHz) for >30minutes. This measure of CBF increased with increasing temperature, and often surpassed the 15Hz limnit at >30tC. Cholinergic stimulation with 10LM ACh produced a reversible increase in CBF at 20tC by -30% above baseline. By switching the light path to an intensified CCD camera, we could measure Fura-2 fluorescence of the same ciliated cell. Using the ratio of emitted light with altemating 340/380nm excitation, we found ACh reversibly increased lCa2%i. This [Ca2+ji increase was likely due to Ca from intemal stores, since Ca-free medium did not prevent the rise in lCa2+li. This demonstrates that [Ca2+1i and CBF can be measured in a single cell and that ACh produces temporally correlated increases in CBF and [Ca2+li. thus setting the stage for an exploration of the role of [Ca2+1i in the regulation of CBF. (Supported in part by the Swiss National Science Foundation). 98195. Deuterium NMR and tim-resolved fluorescence polarization anisorpy (FPA) measurements were made to deternine the rms amplitude of local angula motion of purines in a 12 bp duplex DNA (CGCGAAITCGCG) which is deuterated at the H8 positions of the adenines and guanines. FPA measurements of this sample made in dilute solution yield the hydrodynamic radius of the DNA, RH = 9.94 ± 0.2 A. FPA measurements of the sample at the NMR concentration are employed to ch the collective motions of the DNA in terms of either an enhanced viscosity or end-to-end dimer formation. Expressions we have derived for R2 and the results of the FPA measurements are used to analyze the linewidth of the deuterium NMR spectrum. When the principal-axis frame of the electric field gradient tensor is assumed to undergo overdamped libration around each of its three body-fixed axes in an isotropic deflection potential, then therms amplitude of local motion around any single axis is found to lie in the range 10 to 110 provided the high DNA concentration acts to enhance the viscosity, and about 90 if it acts to produce end-to-end dimers. The proton NMR data of Eimer et al. are manalyzed and shown to yield an rms amplitude of angular motion of the cytosine H5-H6 internuclear vector of 9 to 10°depending upon its orientation with respect to the helix-axis. Within experimental effor these results lie in the same range (8 to 10°) inferrd for base motions at low and intermediate hydration levels in the solid state. Over the past decade, we have reported on a series of spin-labeled nucleic acids containing nitroxide rings attached to thymidine and cytidine bases via tethers of various lengths. The present study focuses on a double-stranded DNA 15mer, 30mer, and 45mer singly-labeled with the 2-atom tethered nitroxide DUMTA . Analysis of the experimental data has been accomplished in a manner similar to that of Hustedt et al.  in their implementation of the model-free approach of Lipari and Szabo (3]. The dynamics are separated into a length dependent global motion arising from overall tumbling and a length independent local motion resulting from base and probe oscillation. The oligonucleotides are modeled as cylinders and the internal motion is accounted for by averaging the g and A magnetic tensors. Currently, we have not employed a model for tensor averaging. Simulations have been fit to the experimental spectra empirically. Results with the DUMTA-labeled oligomers indicate that there is a high degree of tensor averaging indicative of rapid internal motion. This is in contrast to results obtained with a 2-atom acetylenic-tethered nitroxide (ACET) where much less tensor averaging was observed (2]. Molecular dynamics simulations performed with Biosym's InsightIl and Discover software do not indicate a significant difference in mobility between DUMTAand ACET-labeled bases. Proton nuclear magnetic resonance (NMR) spectroscopy is being used to investigate the dependence on salt of the equilibria and kinetics of strand association of the self-complementary dodecamer 5'-d(CGCGAATTCGCG)-3'. The helix-coil transition is monitored using the proton methyl resonances of the two thymines, as a function of the concentration of sodium ions (from 0.05 M to 0.26 M). The equilibrium constant for helix formation (K) is obtained from chemical shift measurements and the rate constants for strand association (ka) and duplex dissociation (kd) from line width measurements. The number of counterions released in the helix-coil transition and their distribution between the association and dissociation processes are calculated from the salt dependence of K., and ka and kd, respectively. The results indicate that the standard enthalpy change (90 ± 10 kcal/mol of duplex) is independent of counterion concentration and, most of the temperature dependence of the equilibrium constant can be attributed to the activation energy for the dissociation process (80 ± 20 keal/mol of duplex). A total of 2.1±0.3 sodium ions are found to be thermodynamically released in the helix-coil transition. Moreover, the number of sodium ions that associate with the DNA during strand association is found to be, within experimental errors, the same as the number of sodium ions released during helix dissociation. The relationship between these results and previous experimental and theoretical findings on this and related DNA oligomers will be discussed. City, School of Basic Life Sciences, Kansas City, Missouri, Differential scanning caloimetry (DSC), laser Raman specopy, pH potentiomety, and optical denio y have bee used to investgaae DNA melting profiles in the presence of Ni2+ and Cd2+. The destabiliing effets of these metaus on the DNA duplex are seen trough lowermeltng empeatues and spectoscopic indications of metal-base binding, decsed backbone order, base unstacking, and ruptured hydrogen bonds. pH potendomety shows that interactons of Ni2+ and Cd2+ with DNA cause a pH drop by more than 2.5 units in wealdy buffered solutions. This effect, which is partially reversible upon DNA melting, provides information about probable metal binding sites. From these results, a mechanism is proposed in which the metal cations enhance the carcinogenic effecs ofcertain com that require acid catalysis for activation. Opdcal densiometry, which was ustodetect DNA aggregation, shows increased turbidit in the range of temperatures where the melting tnsition is detected by Raman spectroscopy and DSC We employ a statistical theory to descibe this phenotnenon in which the configurational entropy of the aggregate is the drving force for DNA aggregation at elevated temperatures in the pesence of divalent metal ions. A 16-mer 5'R-GTAAAACGACGGCCAG-3'F (016), where R is Rhodamine X, and F is fluorescein was prepared for energy tranr studies. When F was excited at 488nm, emission was observed from both the fluorescein and rhodamine. Most of the rhodamine fluorescein derived from fluorescence energy transfer from the fluorescein, and the rato of acceptor fluoecence to donor fluoresence provided a sensitive measure of hybridization of the oligo probe. The 3'-5' distance distribution was explored using steady-state methods with 1as the quencher to vary the FOrster Ro. In IM NaCl, data for the free probe could be fit well to a random coil, whereas upon hybridization, a shifted Gaussian was used. The average (RMS) end-t-end distacs were repectively 36A and 66A. The value of a following hybridization, 17A, appears too large for a rigid helix. The kinetics of hybridization of 016 to the complementary region in M13mpl8(+) followed simple monophasic second-order kinedcs with a rate constant 0.1 that for a 16-mer complement. The kinedcs arm consistent with rapid seoondary structural fluctations around the target site.