Direct observation of defect structure in protein crystals by atomic force and transmission electron microscopy.

We have examined the structure of S-layers isolated from Sulfolobus acidocaldarius using atomic force microscopy (AFM) and transmission electron microscopy (TEM). From the AFM images, we were able to directly observe individual dimers of the crystal, defects in the crystal structure, and twin boundaries. We have identified two types of boundaries, one defined by a mirror plane and the other by a glide plane. This work shows that twin boundaries are highly structured regions that are directly related to the organization of units within each crystal domain. Projection maps from TEM images have shown that there are significant differences in the final average maps has allowed us to relate high magnification views obtained by AFM to the relatively high resolution information obtained by electron microscopy and image processing.     

Direct observation of positive supercoils introduced by reverse gyrase through atomic force microscopy

Reverse gyrase is a hyperthermophilic enzyme that can introduce positive supercoiling in substrate DNA. It is showed in our studies that positive DNA supercoils were induced in both pBR322 vector and an artificially synthesized mini-plasmid DNA by reverse gyrase. The left-handed structures adopted by positively supercoiled DNA molecules could be identified from their right-handed topoisomers through atomic force microscopic examination. Additional structural comparisons revealed that positively supercoiled DNA molecule AFM images exhibited increased contour lengths. Moreover, enzymatic assays showed that the positively supercoiled DNA could not be cleaved by T7 endonuclease. Together, this suggests that the overwound structure of positive supercoils could prevent genomic duplex DNA from randomly forming single-stranded DNA regions and intra-stranded secondary structures.     

Size distribution measurement of vesicles by atomic force microscopy

Vesicles have been utilized as nanoscale vehicles for reagents including potential drug delivery systems. When used to deliver drugs, vesicle size and the size distribution are important factors in the determination of the dosage, cell specificity, and rate of clearance from the body. Current size measurement techniques for vesicles are electron microscopy and dynamic light scattering, but their results are not equal. Therefore atomic force microscopy was attempted as another size measurement technique. After adsorption of the vesicles from a low-concentration solution of vesicles on mica substrate, each vesicle is generally found as a flattened structure. The diameters of vesicles in these solutions and their distribution have been successfully estimated from the surface area of the flattened structure of each vesicle. At higher concentrations, we have found a monolayer crammed with dome-shaped vesicles on the substrate. The diameters of vesicles in these solutions have also been successfully estimated from the surface area of the dome-shaped structure of each vesicle. Diameters of vesicles in solution estimated from two different vesicle concentrations are not close to those reported by electron microscope studies but are close to those reported by dynamic light scattering studies.     

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.     

Direct observation of biopolymers produced from submerged culture of edible mushrooms by atomic force microscopy

Two biopolymers produced from submerged culture of edible mushrooms were directly observed by atomic force microscopy. Biopolymers were deposited on mica from dilute aqueous solution and imaged in air through a thin layer of adsorbed water and their hydrated structures were observed by a tapping mode. A single biopolymer molecule obtained from Cordyceps militaris was typical of a rod-like structure with bending point, which can form intra- and inter-molecular supercoils. In contrast, the image for low molecular weight biopolymer from Paecilomyces sinclarii is typical of a branched structure in which more extensive interaction leads to the formation of network-like matrix.     

Single protein misfolding events captured by atomic force microscopy.

Using single protein atomic force microscopy (AFM) techniques we demonstrate that after repeated mechanical extension/relaxation cycles, tandem modular proteins can misfold into a structure formed by two neighboring modules. The misfolding is fully reversible and alters the mechanical topology of the modules while it is about as stable as the original fold. Our results show that modular proteins can assume a novel misfolded state and demonstrate that AFM is able to capture, in real time, rare misfolding events at the level of a single protein.     

Direct visualization of polypeptide shell of ferritin molecule by atomic force microscopy.

The polypeptide shell of the ferritin molecule has been imaged in water by atomic force microscopy (AFM). The central dip and the quaternary structure could be observed on the surface of the ferritin molecule anchored inhomogeneously in two dimensions. These structures observed in the AFM images are quite similar to the electron density map near the top of the apoferritin viewed down from a 4-fold axis structure reported previously (S. H. Banyard, D. K. Stammers, and P. M. Harrison, 1978. Nature (Lond.). 271:282-284). It has been achieved by introducing a "self-screening effect" of the surface charges of the AFM sample (S. Ohnishi, M. Hara, T. Furuno, and H. Sasabe. 1992. Biophys. J. 63:1425-1431) and the specially sharpened stylus of AFM cantilever.     

Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy

Using a new scheme based on atomic force microscopy (AFM), we investigate mechanical properties of clathrin-coated vesicles (CCVs). CCVs are multicomponent protein and lipid complexes of approximately 100 nm diameter that are implicated in many essential cell-trafficking processes. Our AFM imaging resolves clathrin lattice polygons and provides height deformation in quantitative response to AFM-substrate compression force. We model CCVs as multilayered elastic spherical shells and, from AFM measurements, estimate their bending rigidity to be 285 +/- 30 k(B)T, i.e., approximately 20 times that of either the outer clathrin cage or inner vesicle membrane. Further analysis reveals a flexible coupling between the clathrin coat and the membrane, a structural property whose modulation may affect vesicle biogenesis and cellular function.     

The structure and stability of phospholipid bilayers by atomic force microscopy.

Atomic force microscopy (AFM) was used to investigate the structure, stability, and defects of the hydrophilic surfaces of Langmuir-Blodgett bilayer films of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylethanolamine (DPPE) in the solid phase, and dilinoleoylphosphatidylethanolamine (DLPE) in the fluid phase. Their relative resilience to external mechanical stress by the scanning tip and by fluid exchange were also investigated. DPPE monolayers showed parallel ridges at the surface with a period of 0.49 nm, corresponding to the rows of aligned headgroups consistent with the known crystallographic structure. DSPC and DLPE monolayers did not show any periodic order. The solid DSPC and DPPE monolayers were stable to continued rastering by the AFM tip; however, the stability of DLPE monolayers depended on the pH of the aqueous environment. Structural defects in the form of monolayer gaps and holes were observed after fluid exchange, but the defects in DLPE monolayer at pH 11 were stable during consecutive scanning. At pH 9 and below, the defects induced by fluid exchange over DLPE monolayers were more extensive and were deformed easily by consecutive scanning of the AFM tip at a force of 10 nN. The pH dependence of resilience was explained by the increasing bending energy or frustration due to the high spontaneous curvature of DLPE monolayers at low pH. The tangential stress exerted by the AFM tip on the deformable monolayers eventually produced a ripple pattern, which could be described as a periodic buckling known as Shallamach waves.     

Subfibrillar structure of type I collagen observed by atomic force microscopy.

We have imaged native rat tail and reconstituted bovine dermal type I collagen by atomic force microscopy, obtaining a level of detail comparable to that obtained on the same samples by transmission electron microscopy. The characteristic 60-70 nm D periodicity consists of ridges exhibiting high tip-sample adhesion alternating with 5-15-nm-deep grooves having low adhesion. We also observe an intraperiod or "minor" band consisting of 1-nm-deep grooves, and "microfibrils" arranged parallel to or inclined approximately 5 degrees to the fibril axis. In air collagen fibrils exhibit negligible compression under the forces exerted by the tip. When immersed in water the subfibrillar features disappear and the fibrils become softer, compressing by 5% of their height under an 11-nN force. Material on the surface of the sample sometimes accumulates on the atomic force microscope tip; contrary to expectation such tip contamination can improve as well as reduce resolution.     

Hyaluronic acid by atomic force microscopy.

Hyaluronic acid (HA) of different molecular weights has been examined by atomic force microscopy (AFM) in air. This technique allows 3-D surface images of soft samples without any pretreatment, such as shadowing or staining. In the present study we examined the supermolecular organization of HA chains when deposited on mica and graphite, to better understand the interchain and intrachain interactions of HA molecules in solution. The concentration of the solution deposited varied from 0.001 to 1 mg/ml. On both substrates, and independent of the concentration, high-molecular-mass HA formed networks in which molecules ran parallel for hundreds of nanometers, giving rise to flat sheets and tubular structures that separate and rejoin into similar neighboring aggregates. Accurate measurements of the thickness of the thinnest sheets were consistent with a monolayer of HA molecules, 0.3 nm thick, strongly indicating lateral aggregation forces between chains as well as rather strong hydrophilic interactions between mica and HA. The results agree with an existing model of HA tertiary structure in solution in which the network is stabilized by both hydrophilic and hydrophobic interactions. Our images support this model and indicate that hydrophobic interactions between chains may exert a pivotal role in aqueous solution.     

Direct observation of poly(3-hydroxybutyrate) depolymerase adsorbed on polyester thin film by atomic force microscopy

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerases adsorbed on poly(L-lactide) (PLLA) thin film were directly observed by atomic force microscopy (AFM). A PLLA thin film of 100 nm thickness was prepared on a silicon wafer by spin-cast method. The PLLA thin film was treated at 220 degrees C and quenched to room temperature, resulting in the formation of a completely amorphous film with a smooth surface. Then, the PHB depolymerases from Pseudomonas stutzeri YM1006 and Ralstonia pickettii T1 were dispersed on the amorphous PLLA thin film. Direct AFM observation has revealed that the PHB depolymerases bind in an elliptic shape on the surface of the PLLA thin film and that a small ridge is created around each enzyme molecule. After removal of the enzymes with 40% ethanol aqueous solution, small hollows were found on the PLLA thin film. These results suggest that a PHB depolymerase interacts with polyester molecules during their adsorption to make a hollow on the substrate surface.     

New internal structure of spider dragline silk revealed by atomic force microscopy.

Atomic force microscopy was used to study the three-dimensional nanometer-scale structure of the dragline silk of Nephila clavipes from microtomed sections of the silk. Contrary to a previously proposed model of randomly distributed protein crystallites interspersed in amorphous regions, a highly organized skin-core structure of the fiber was observed. The skin appeared to be thin with no discernible distinct features. The core consists of pleated fibril-like structures, which are arranged in two concentric cylinders. Upon stretching, the pleats were smoothed out substantially. The mechanical properties of spider silk can quite straightforwardly be related to the newly observed structures.     

Direct visualization of KirBac3.1 potassium channel gating by atomic force microscopy

KirBac3.1 belongs to a family of transmembrane potassium (K⁺) channels that permit the selective flow of K-ions across biological membranes and thereby regulate cell excitability. They are crucial for a wide range of biological processes and mutations in their genes cause multiple human diseases. Opening and closing (gating) of Kir channels may occur spontaneously but is modulated by numerous intracellular ligands that bind to the channel itself. These include lipids (such as PIP₂), G-proteins, nucleotides (such as ATP) and ions (e.g. H⁺, Mg²⁺, Ca²⁺). We have used high-resolution atomic force microscopy (AFM) to examine KirBac3.1 in two different configurations. AFM imaging of the cytoplasmic surface of KirBac3.1 embedded in a lipid bilayer has allowed visualization of the tetrameric assembly of the ligand-binding domain. In the absence of Mg²⁺, the four subunits appeared as four protrusions surrounding a central depression corresponding to the cytoplasmic pore. They did not display 4-fold symmetry, but formed a dimer-of-dimers with 2-fold symmetry. Upon addition of Mg²⁺, a marked rearrangement of the intracellular ligand-binding domains was observed: the four protrusions condensed into a single protrusion per tetramer, and there was an accompanying increase in protrusion height. The central cavity within the four intracellular domains also disappeared on addition of Mg²⁺, indicating constriction of the cytoplasmic pore. These structural changes are likely transduced to the transmembrane helices, which gate the K⁺ channel. This is the first time AFM has been used as an interactive tool to study K⁺ channels. It has enabled us to directly measure the conformational changes in the protein surface produced by ligand binding.     

Imaging biomolecule arrays by atomic force microscopy.

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.     

Preparation of DNA catenanes and observation of their topological structures by atomic force microscopy.

DNA catenanes have been prepared by the reaction of T4 DNA ligase with linear DNA in the presence of nicked DNA. Single molecular images of DNA catenanes and large circular DNAs have been clearly observed by AFM using a tapping mode at room temperature and in an ambient atmosphere.     

Direct aldosterone action on mouse cardiomyocytes detected with atomic force microscopy.

There is growing evidence that aldosterone acts on heart where it causes cellular remodeling and hypertrophy. Since it is still unclear whether aldosterone directly acts on cardiomyocytes or indirectly, by an altered electrolyte balance in the organism, we applied atomic force microscopy (AFM) in primary cultures of neonatal mouse cardiomyocytes to measure hormone-induced changes in cell volume and plasma membrane surface. AFM measures cell volume and, at the same time, provides quantitative information on cell surface properties. Neonatal mouse cardiomyocytes were cultured for 28 hours in absence or presence of 100 nM aldosterone. Spironolactone was applied as a selective aldosterone receptor antagonist. At the microscopic level, single cell volume and single cell surface were found unchanged by aldosterone. However, nanoscopy of the cell surface, i.e. analysis of the plasma membrane at the nanometer level, revealed a specific increase in plasma membrane nano-enfoldings (roughness). This aldosterone-mediated increase in cell surface roughness was completely prevented by spironolactone. We conclude: (i) Aldosterone directly acts upon cardiomyocytes. (ii) At the microscopic level, no changes of cell volume and cell surface are detectable. (iii) At the nanoscopic level, aldosterone increases plasma membrane roughness. These nanometer changes, detectable only with AFM in cells scanned in fluid after fixation under physiological conditions, indicate plasma membrane remodeling of cardiomyocytes by mineralocorticoids.     

Localization of the lipopolysaccharide-binding protein in phospholipid membranes by atomic force microscopy

Lipopolysaccharides (LPS; endotoxin) activate immunocompetent cells of the host via a transmembrane signaling process. In this study, we investigated the function of the LPS-binding protein (LBP) in this process. The cytoplasmic membrane of the cells was mimicked by lipid liposomes adsorbed on mica, and the lateral organization of LBP in these membranes and its interaction with LPS aggregates were characterized by atomic force microscopy. Using cantilever tips functionalized with anti-LBP antibodies, single LBP molecules were localized in the membrane at low concentrations. At higher concentrations, LBP formed clusters of several molecules and caused cross-linking of lipid bilayers. The addition of LPS to LBP-containing liposomes led to the formation of LPS domains in the membranes, which could be inhibited by anti-LBP antibodies. Thus, LBP mediates the fusion of lipid membranes and LPS aggregates.