Structural Changes in Tubulin Sheets Caused by Immobilization on Solid Supports.

In the presence of zinc, the protein tubulin assembles into two-dimensional sheets that are a useful model system for the study of both tubulin and microtubule structure. Tubulin sheets present an ideal protein structure for study with atomic force microscopy because they contain a two-dimensional crystalline protein lattice and retain many of the structural features of tubulin and microtubules. However, high-resolution imaging requires nonperturbative immobilization onto an appropriate imaging substrate. In this report, several substrates commonly used for scanning probe microscopy are evaluated for their ability to effectively immobilize tubulin sheets: mica, gold, highly ordered pyrolytic graphite, and carbon-coated electron microscopy grids. We hypothesize that the different intermolecular interactions presented by these substrates will affect the morphology of adsorbed tubulin sheets as well as the amount of other contaminating adsorbates. Tubulin sheets were successfully imaged on all of these substrates and structural characterization is reported. The most consistent results were obtained on carbon-coated electron microscopy grids, which preserved fine structural features of the sheets and had the least amount of contamination from the adsorption of unpolymerized tubulin. Images of tubulin sheets obtained with atomic force microscopy also compare favorably with published electron micrographs of sheets produced using similar procedures. This work demonstrates the importance of assessing substrate effects when studying two-dimensional protein crystals and identifies suitable substrates for immobilizing tubulin sheets.

Dimerization and Long-Range Repulsion Established by Both Termini of the Microtubule-Associated Protein Tau.

Tau is a microtubule-associated protein found in neuronal axons that has several well-known functions, such as promoting microtubule polymerization, stabilizing microtubules against depolymerization, and spatially organizing microtubules in axons. Two contrasting models have been previously described to explain tau's ability to organize the spacing between microtubules: complementary dimerization of the projection domains of taus on adjacent microtubules or tau's projection domain acting as a polyelectrolyte brush. In this study, atomic force microscopy was used to interrogate intermolecular interactions between layers of tau protein immobilized on mica substrates and on silicon nitride atomic force microscope tips. On these surfaces, tau adopts an orientation comparable to that when bound to microtubules, with the basic microtubule binding domain immobilized and the acidic domains extending into solution. Force-distance curves collected via atomic force microscopy reveal that full length human tau, when assembled into dense surface-bound layers, can participate in attractive electrostatic interactions consistent with the previously reported dimerization model. However, modulating the ionic strength of the surrounding solution can change the structure of these layers to produce purely repulsive interactions consistent with a polyelectrolyte brush structure, thus providing biophysical evidence to support both the zipper and brush models. In addition, a pair of projection domain deletion mutants were examined to investigate whether the projection domain of the protein is essential for the dimerization and brush models. Force-distance curves collected on layers of these proteins demonstrate that the C-terminus can play a role analogous to that of the projection domain.

Mechanics of microtubules: effects of protofilament orientation.

Microtubules are hollow cylindrical polymers of the protein tubulin that play a number of important dynamic and structural roles in eukaryotic cells. Both in vivo and in vitro microtubules can exist in several possible configurations, differing in the number of protofilaments, helical rise of tubulin dimers, and protofilament skew angle with respect to the main tube axis. Here, finite element modeling is applied to examine the mechanical response of several known microtubule types when subjected to radial deformation. The data presented here provide an important insight into microtubule stiffness and reveal that protofilament orientation does not affect radial stiffness. Rather, stiffness is primarily dependent on the effective Young's modulus of the polymerized material and the effective radius of the microtubule. These results are also directly correlated to atomic force microscopy nanoindentation measurements to allow a more detailed interpretation of previous experiments. When combined with experimental data that show a significant difference between microtubules stabilized with a slowly hydrolyzable GTP analog and microtubules stabilized with paclitaxel, the finite element data suggest that paclitaxel increases the overall radial flexibility of the microtubule wall.

Enhanced mechanical stability of microtubules polymerized with a slowly hydrolyzable nucleotide analogue.

Atomic force microscopy (AFM) has been used to investigate the local mechanical and structural properties of microtubules polymerized using guanylyl-alpha-beta-methylene diphosphonate (GMPCPP), a slowly hydrolyzable analogue of guanosine triphosphate. Using a combination of AFM imaging and local force spectroscopy, GMPCPP-polymerized microtubules have been qualitatively and quantitatively compared to paclitaxel-stabilized microtubules. GMPCPP-polymerized microtubules qualitatively display a greater resistance to destruction by the AFM probe tip during imaging and during deformation measurements and maintain structural details after indentation. In addition, using force spectroscopy taken during the indentation and collapse of individual microtubules with the AFM probe tip, an effective spring constant of the microtubule wall (kMT) for both types of microtubules was determined. The average kMT of GMPCPP-polymerized microtubules, 0.172 N/m, is more than twice that of paclitaxel-stabilized microtubules. These results complement previously reported measurements of bending experiments on GMPCPP-polymerized and paclitaxel-stabilized microtubules.

Cross-step place-exchange of oligo(phenylene-ethynylene) molecules.

We have observed nitro-functionalized oligo(phenylene-ethynylene) molecules exhibiting motion up and down Au{111} substrate monatomic step edges within host self-assembled monolayers of n-alkanethiols, independent of previously observed conductance switching. Single molecules have been imaged with scanning tunneling microscopy to place-exchange reversibly between the top and bottom of monatomic substrate step edges.

Substrate-mediated intermolecular interactions: a quantitative single molecule analysis.

Long-range intermolecular interactions mediated by the surface are believed to be responsible for many effects in surface science, including molecular ordering, formation of nanostructures, and aligning reactive intermediates in catalysis. Here, we use scanning tunneling microscopy to probe the weak substrate-mediated interactions in benzene overlayers on Au{111} at 4 K. Using an automated procedure to monitor single molecule motion, we are able to quantify the substrate-mediated interaction strength. We explain quantitatively both the kinetics of the benzene motion and the thermodynamics that determine the packing structures benzene adopts in this system in light of these substrate-mediated interactions.

Benzene on Au[111] at 4 K: monolayer growth and tip-induced molecular cascades.

Low-temperature scanning tunneling microscopy has been used to characterize the various structures of submonolayer and near-monolayer coverages of benzene (C6H6) on Au[111] at 4 K. At low coverage, benzene is found to adsorb preferentially at the top of the Au monatomic steps and is weakly adsorbed on the terraces. At near-monolayer coverage, benzene was found to form several long-range commensurate overlayer structures that depend on the regions of the reconstructed Au[111] surface, namely a (radical 52 x radical 52)R13.9 degrees structure over the hcp regions and a (radical 133 x radical 133)R17.5 degrees "pinwheel" structure over the fcc regions. Time-lapse imaging revealed concerted cascade motion of the benzene molecules in the (radical 133 x radical 133)R17.5 degrees pinwheel overlayer. We demonstrate that the observed cascade motion is a result of concerted molecular motion and not independent random motion.

Control of alkanethiolate monolayer structure using vapor-phase annealing.

We describe an annealing procedure for self-assembled monolayers (SAMs) that uses vapor-phase molecules to modify the local domain structure. Existing SAMs of decanethiolate on Au{111} were annealed using vapor-phase dodecanethiol molecules, so that the original and newly introduced molecules could be distinguished using scanning tunneling microscopy (STM). Molecules deposited from the vapor phase inserted at existing monolayer defect sites and domain boundaries, and at substrate step edges forming discrete network-like domains. The SAM molecular lattice can be preserved across molecular terrace boundaries between the decanethiolate and dodecanethiolate domains. Candidate molecular electronic component molecules were inserted from solution in the decanethiolate matrix as isolated molecules. These inserted molecules could then be surrounded by dodecanethiolate molecules introduced from the vapor phase, thus demonstrating a method for controlling the local environment of inserted molecules.