Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Visualization of plant cell walls by atomic force microscopy.

Atomic force microscopy has been used to visualize the ultrastructure of hydrated plant cell wall material from prepared apple (Malus pumila MILL; Cox orange pippin), water chestnut (Eleocharis dulcis L.), potato (Solanum tuberosum L.; Bintje), and carrot (Daucus carota L.; Amsterdamse bak) parenchyma. Samples of cell wall material in aqueous suspension were deposited onto freshly cleaved mica. Excess water was blotted away and the moist samples were imaged in air at ambient temperature and humidity. The three-dimensional images obtained highlighted the layered structure of the plant cell walls and revealed features interpreted as individual cellulose microfibrils and plasmodesmata.     

Visualization of intrinsically disordered proteins by high-speed atomic force microscopy

High-speed atomic force microscopy (HS-AFM) is a powerful tool established 13 years ago. This methodology can capture individual protein molecules carrying out functional activities under near-physiological conditions, without chemical labeling, at 2–3 nm lateral and ∼0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution. Although most biological HS-AFM studies thus far target structured proteins, HS-AFM is also ideally suited to study the dynamics of intrinsically disordered proteins. Here we review some of the dynamic structures and processes of intrinsically disordered proteins that have been unveiled by HS-AFM imaging.     

Energy landscape of streptavidin-biotin complexes measured by atomic force microscopy

The dissociation of ligand and receptor involves multiple transitions between intermediate states formed during the unbinding process. In this paper, we explored the energy landscape of the streptavidin-biotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand-receptor complexes. The rupture force of the streptavidin-biotin bond increased more than 2-fold over a range of loading rates between 100 and 5000 pN/s. Moreover, the force measurements showed two regimes of loading in the streptavidin-biotin force spectrum, revealing the presence of two activation barriers in the unbinding process. Parallel experiments carried out with a streptavidin mutant (W120F) were used to investigate the molecular determinants of the activation barriers. From these experiments, we attributed the outer activation barrier in the energy landscape to the molecular interaction of the '3-4' loop of streptavidin that closes behind biotin.     

RecA-double stranded DNA complexes studied by atomic force microscopy.

RecA-double stranded (ds) DNA complexes have been studied by atomic force microscopy (AFM). When the complexes were prepared in the presence of ATP gamma S, fully covered RecA-dsDNA filaments were observed by AFM. When the concentration of RecA proteins was lower, various lengths of filaments were found. The variation of the observed structures may directly reflect the real distribution of the intermediate complexes in the reaction mixture, as the mixture was simply deposited on a mica surface for AFM observation without special fixation or staining. The use of a carbon nanotube (CNT) AFM tip enabled high resolution to reveal the periodicity of RecA-dsDNA filaments. Our observations demonstrated the potential of the AFM method for the structural studies of the RecA-dsDNA complexes, especially their intermediate states.     

Detection of immune complexes using atomic force microscopy

Complex formation between immunoglobulins and ligands immobilized on mica was studied by atomic force microscopy in two different systems. In the first system, 60-kDa ligands possessing only one site for antibody recognition were used. In the other system, a more complex interaction of human immunoglobulin with immobilized polyclonal antibodies was studied. In both systems, specific complexes with proper ligand appeared, and unspecific interaction was not detected. The method of revealing immunocomplexes by image atomic force microscopy can be used in the development of modern diagnostic systems.     

Visualization and characterization of prolamellar bodies with atomic force microscopy

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1–2 μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1 μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25 min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl₂ also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.     

Visualization of the cruciform structure of superhelical DNA by use of atomic force microscopy

Atomic force microscopy was used to visualize the cruciform structure in supercoiled plasmid pUC8 DNA immobilized on aminomodified mica. The cruciform hairpin was 14 base pairs in size, as determined from atomic force microscopy images of pUC8 DNA in air. Molecular modeling confirmed that the cruciform structure is formed by hairpins with self-complementary homopyrimidine-homopurine sequences (dT)8(dA)6 and a loop 4 nucleotides long.     

Differentiating inclusion complexes from host molecules by tapping-mode atomic force microscopy.

Tapping-mode atomic force microscopy imaging under different cantilever vibration amplitudes has been used to differentiate the host beta-cyclodextrin nanotubes from retinal/beta-cyclodextrin inclusion complex nanotubes. It was observed that both compounds were deformed differently by the applied probe force because of their different local rigidity. This change in the elasticity properties can be explained as a consequence of the inclusion process. This method shows that tapping-mode atomic force microscopy is an useful tool to map soft sample elasticity properties and to distinguish inclusion complexes from their host molecules on the basis of their different mechanical response.     

High-resolution atomic force microscopy visualization of metalloproteins and their complexes

Background

Metalloproteins myeloperoxidase (MPO), ceruloplasmin (CP) and lactoferrin (LF) play an important role in regulation of inflammation and oxidative stress in vertebrates. It was previously shown that these proteins may work synergetically as antimicrobial and anti-inflammatory agents by forming complexes, such as MPO-CP and LF-CP. However, interaction of metalloprotein molecules with each other has never been characterized at a single-molecule level.

Imaging polysaccharides by atomic force microscopy

Techniques have been developed for the routine reliable imaging of polysaccharides by atomic force microscopy (AFM). The polysaccharides are deposited from aqueous solution onto the surface of freshly cleaved mica, air dried, and then imaged under alcohols. The rationale behind the development of the methodology is described and data is presented for the bacterial polysaccharides xanthan, acetan, and the plant polysaccharides 1-carrageenan and pectin. Studies on uncoated polysaccharides have demonstrated the improved resolution achievable when compared to more traditional metal-coated samples or replicas. For acetan the present methodology has permitted imaging of the helical structure. Finally, in addition to data obtained on individual polysaccharides, AFM images have also been obtained of the network structures formed by κ-carrageenan and gellan gum. © 1996 John Wiley & Sons, Inc.     

Metastable and stable states of xanthan polyelectrolyte complexes studied by atomic force microscopy

The compaction of the semiflexible polysaccharide xanthan with selected multi- and polyvalent cations was studied. Polyelectrolyte complexes prepared at concentrations of 1-2 microg/ml were observed by tapping mode atomic force microscopy. High-molecular-weight xanthan compacted with chitosan yields a blend of mainly toroidal and metastable structures and a small fraction of rod-like species. Polyelectrolyte complexes of xanthan with polyethylenimine and trivalent chromium yielded similar structures or alternatively less well packed species. Racquet-type morphologies were identified as kinetically trapped states occurring on the folding path toward the energetically stable state of the toroids. Thermal annealing yielded a shift of the distribution of xanthan-chitosan morphologies toward this stable state. Ensembles of toroidal and rod-like morphologies of the xanthan-chitosan structures, collected using an asphericity index, were analyzed. The mean height of the toroids increased upon heating, with a selective increase in the height range above 2 nm. It is suggested that the observed metastable structures are formed from the high-molecular-weight fraction of xanthan and that these are driven toward the toroidal state, being a low-energy state, following annealing. Considered a model system for condensation of semiflexible polymers, the compaction of xanthan by chitosan captures the system at various stages in the folding toward a low-energy state and thus allows experimental analyses of these intermediates and their evolution.     

Visualization of single Escherichia coli FtsZ filament dynamics with atomic force microscopy

FtsZ, the prokaryotic homologue of tubulin, is an essential cell division protein. In the cell, it localizes at the center, forming a ring that constricts during division. In vitro, it binds and hydrolyzes GTP and polymerizes in a GTP-dependent manner. We have used atomic force microscopy to study the structure and dynamics of FtsZ polymer assembly on a mica surface under buffer solution. The polymers were highly dynamic and flexible, and they continuously rearranged over the surface. End-to-end joining of filaments and depolymerization from internal zones were observed, suggesting that fragmentation and reannealing may contribute significantly to the dynamics of FtsZ assembly. The shape evolution of the restructured polymers manifested a strong inherent tendency to curve. Polymers formed in the presence of non-hydrolyzable nucleotide analogues or in the presence of GDP and AlF(3) were structurally similar but showed a slower dynamic behavior. These results provide experimental evidence supporting the model of single-strand polymerization plus cyclization recently proposed to explain the hydrodynamic behavior of the polymers in solution.