Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy.

Samples of supported planar lipid-protein membranes and actin filaments on mica were imaged by atomic force microscopy (AFM). The samples were fully submerged in buffer at room temperature during imaging. Individual proteins bound to the reconstituted membrane were distinguishable; some structural details could be resolved. Also, surface-induced, self-assembling of actin filaments on mica could be observed. Monomeric subunits were imaged on individual actin filaments. The filaments could be manipulated on or removed from the surface by the tip of the AFM. The process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was imaged in real time.     

A COOH-terminal peptide confers regiospecific orientation and facilitates atomic force microscopy of an IgG1.

An antibody (IgG1) was designed for oriented adherence to a metal-containing surface. This was achieved by adding a metal-chelating peptide, (CP = His-Trp-His-His-His-Pro), to the COOH-terminus of the heavy chain through genetic engineering. Electroporation of the engineered heavy chain gene into cells expressing the complimentary light chain yielded colonies secreting an intact antibody containing the metal-chelating peptide (IgG1-CP) which had high affinity for a nickel-loaded iminodiacetate column. Purified IgG1-CP was bound to nickel-treated mica and imaged by atomic force microscopy (AFM). Antibody lacking the COOH-terminal metal binding peptide failed to produce discernible AFM images. The AFM images of individual IgG1-CP molecules and their calculated dimensions demonstrated that regiospecific binding and uniform orientation of the antibody was imparted by the peptide. The ability to stably orient macromolecules in their native state to a surface may be used advantageously to visualize them.     

Atomic force microscopy of liposomes bearing fibrinogen

Extruded liposomes formed from dipalmitoylphosphatidylcholine and cholesterol, with and without fibrinogen, were examined by atomic force microscopy (AFM). The sequence of events involved in the transition from attached liposomes to bilayer patches on mica supports was viewed by tapping mode in liquid. After adhesion to the mica surface, both liposomes without fibrinogen and liposomes with attached fibrinogen collapsed into patches. The fibrinogen layer attached to the liposomes was 2.6 nm thick. This implied that the protein was spread over the entire liposome and the protein characteristic trinodular structure disappeared. To check the type of bond between fibrinogen and liposome, sequential images were taken after the incubation of fibrinogen with liposomes with and without a chemical group for attaching the protein. The results clearly confirmed that fibrinogen bound covalently to liposomes.     

Conformational dynamics of individual antibodies using computational docking and AFM

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. In this study, we aimed at analyzing the conformational variability of receptors portrayed by monoclonal antibodies that have been individually imaged using atomic force microscopy (AFM). Individual antibodies were chemically coupled to activated mica surface, and they have been imaged using AFM in ambient conditions. The resulting topographical surface of antibodies was used to assemble the three subunits constituting antibodies: two antigen‐binding fragments and one crystallizable fragment using a surface‐constrained computational docking approach. Reconstructed structures based on 10 individual topographical surfaces of antibodies are presented for which separation and relative orientation of the subunits were measured. When compared with three X‐ray structures of antibodies present in the protein data bank database, results indicate that several arrangements of the reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X‐ray structure consequence of the antibody flexibility. We conclude that high‐resolution AFM imaging with appropriate computational reconstruction tools is adapted to study the conformational dynamics of large individual macromolecules deposited on mica. Copyright © 2013 John Wiley & Sons, Ltd.     

Direct sensing of ligand-protein interaction by counting of bioaffinity beads with atomic force microscopy

Estradiol-displayed bioaffinity beads binding to the anti-estradiol antibody attached via the protein A-coated mica surface were examined by atomic force microscopy (AFM). The amount of specifically bound beads on the surface was directly proportional to the concentration of free estradiol in solution. Estradiol from 10 ng ml^–1 to 10 g ml^–1 could be determined. This suggested that direct counting of bioaffnity beads by AFM can be used to detect specific ligand for the target protein.     

Study of microorganism genome DNA by atomic force microscopy

The potential of atomic force microscopy (AFM) for the investigation of peculiarities of microorganisms genome structure is demonstrated. AFM images of phage lambda DNA linear molecules and supercoiled mica in buffer solution was imaged in air. New experimental method of DNA stretching based on using amino-modified mica with a decreased surface density of active amino-groups is proposed. Stretched molecules of phage lambda DNA were imaged by AFM.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

Monoclonal antibody covalently coupled to liposomes: Specific targeting to cells

We have evaluated optimal conditions for coupling monoclonal antibody to small unilamellar lipisomes. Coupling of an IgG_2a monoclonal anti-β₂-microglobulin antibody, which reacts with human cells, was examined in detail. Liposomes were composed of dipalmitoyl lecithin and cholesterol, and variable quantities of phosphatidylethanolamine substituted with the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio) propionate (SPDP). They were reacted with antibody derivatized with the same reagent at a 5- to 20-fold molar excess, and activated by mild reduction. This degree of SPDP modification had no effect on the capacity of the antibody to bind to its target antigen. More than 40% of antibody could be reproducibly bound to liposomes, resulting in the coupling of from 1 to 10 antibody molecules per liposome (mean diameter.580 Å). The coupling reaction did not lead to loss of carboxyfluorescein encapsulated within liposomes. At least 80% of liposomes carried nondenatured antibody, as confirmed by precipitation of liposomes and encapsulated carboxyfluorescein by Staphylococcus aureus, strain Cowan I. The liposome-coupled antibody retained its immunological specificity: only cells expressing human β₂-microglobulin bound liposomes in vitro, and the binding was inhibited by the free antibody in solution. Results with antibodies of different antigenic specificity confirm that the technique can be generally applied.     

Hyaluronan conformations on surfaces: effect of surface charge and hydrophobicity

Extended, relaxed, condensed, and interacting forms of the polysaccharide hyaluronan have been observed by atomic force microscopy (AFM). The types of images obtained depend on the properties of the surfaces used. We have investigated several different surface conditions for HA imaging, including unmodified mica, mica chemically modified with two different kinds of amino-terminated silanes (3-aminopropyltriethoxysilane and N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride), and highly oriented pyrolytic graphite. We found the degree of HA molecular extension or condensation to be variable, and the number of bound chains per unit area was low, for all of the mica-based surfaces. HA was more easily imaged on graphite, a hydrophobic surface. Chains were frequently observed in high degrees of extension, maintained by favorable interaction with the surface after molecular combing. This observation suggests that the HA macromolecule interacts with graphite through hydrophobic patches along its surface. AFM studies of HA behavior on differing surfaces under well-controlled environmental conditions provides useful insight into the variety of conformations and interactions likely to be found under differing physiological conditions.     

Study of the DNA/ethidium bromide interactions on mica surface by atomic force microscope: influence of the surface friction

The influence of mica surface on DNA/ethidium bromide interactions is investigated by atomic force microscopy (AFM). We describe the diffusion mechanism of a DNA molecule on a mica surface by using a simple analytical model. It appears that the DNA diffusion on a mica surface is limited by the surface friction due to the counterion correlations between the divalent counterions condensed on both mica and DNA surfaces. We also study the structural changes of linear DNA adsorbed on mica upon ethidium bromide binding by AFM. It turns out that linear DNA molecules adsorbed on a mica surface are unable to relieve the topological constraint upon ethidium bromide binding. In particular, strongly adsorbed molecules tend to be highly entangled, while loosely bound DNA molecules appear more extended with very few crossovers. Adsorbed DNA molecules cannot move freely on the surface because of the surface friction. Therefore, the topological constraint increases due to the ethidium bromide binding. Moreover, we show that ethidium bromide has a lower affinity for strongly bound molecules due to the topological constraint induced by the surface friction.     

Atomic-force microscopy imaging of plasma membranes purified from spinach leaves

Summary Plasma membranes purified from spinach leaves by aqueous two-phase partitioning were examined by atomic-force microscopy (AFM) in phosphate buffer, and details on their structure were reported at nanometric scale. Examination of the fresh membrane preparation deposited on mica revealed a complex organization of the surface. It appeared composed of a first layer of material, about 8 nm in thickness, that practically covered all the mica surface and on which stand structures highly heterogeneous in shape and size. High-resolution imaging showed that the surface of the first layer appeared relatively smooth in some regions, whereas different characteristic features were observed in other regions. They consisted of globular-to-elliptical protruding particles of various sizes, from 4–5 nm x-y size for the smallest to 40–70 nm for the largest, and of channel-like structures 25–30 nm in diameter with a central hole. Macromolecular assemblies of protruding particles of various shapes were imaged. Addition of the proteolytic enzyme pronase led to a net roughness decrease in regions covered with particles, indicating their proteinaceous nature. The results open fascinating perspectives in the investigation of membrane surfaces in plant cells with the possibility to get structural information at the nanometric range.Abbreviations AFM atomic-force microscopy - EM electron microscopy - TMAFM tapping-mode atomic-force microscopy     

DNA binding to mica correlates with cationic radius: assay by atomic force microscopy.

In buffers containing selected transition metal salts, DNA binds to mica tightly enough to be directly imaged in the buffer in the atomic force microscope (AFM, also known as scanning force microscope). The binding of DNA to mica, as measured by AFM-imaging, is correlated with the radius of the transition metal cation. The transition metal cations that effectively bind DNA to mica are Ni(II), Co(II), and Zn(II), which have ionic radii from 0.69 to 0.74 A. In Mn(II), ionic radius 0.82 A, DNA binds weakly to mica. In Cd(II) and Hg(II), respective ionic radii of 0.97 and 1.1 A, DNA does not bind to mica well enough to be imaged with the AFM. These results may to relate to how large a cation can fit into the cavities above the recessed hydroxyl groups in the mica lattice, although hypotheses based on hydrated ionic radii cannot be ruled out. The dependence of DNA binding on the concentrations of the cations Ni(II), Co(II), or Zn(II) shows maximal DNA binding at approximately 1-mM cation. Mg(II) does not bind DNA tightly enough to mica for AFM imaging. Mg(II) is a Group 2 cation with an ionic radius similar to that of Ni(II). Ni(II), Co(II), and Zn(II) have anomalously high enthalpies of hydration that may relate to their ability to bind DNA to mica. This AFM assay for DNA binding to mica has potential applications for assaying the binding of other polymers to mica and other flat surfaces.     

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.     

Atomic force microscopy of DNA in aqueous solutions.

DNA on mica can be imaged in the atomic force microscope (AFM) in water or in some buffers if the sample has first been dehydrated thoroughly with propanol or by baking in vacuum and if the sample is imaged with a tip that has been deposited in the scanning electron microscope (SEM). Without adequate dehydration or with an unmodified tip, the DNA is scraped off the substrate by AFM-imaging in aqueous solutions. The measured heights and widths of DNA are larger in aqueous solutions than in propanol. The measured lengths of DNA molecules are the same in propanol and in aqueous solutions and correspond to the base spacing for B-DNA, the hydrated form of DNA; when the DNA is again imaged in propanol after buffer, however, it shortens to the length expected for dehydrated A-DNA. Other results include the imaging of E. coli RNA polymerase bound to DNA in a propanol-water mixture and the observation that washing samples in the AFM is an effective way of disaggregating salt-DNA complexes. The ability to image DNA in aqueous solutions has potential applications for observing processes involving DNA in the AFM.     

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.     

Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

Bacterial Immobilization for Imaging by Atomic Force Microscopy

AFM is a high-resolution (nm scale) imaging tool that mechanically probes a surface. It has the ability to image cells and biomolecules, in a liquid environment, without the need to chemically treat the sample. In order to accomplish this goal, the sample must sufficiently adhere to the mounting surface to prevent removal by forces exerted by the scanning AFM cantilever tip. In many instances, successful imaging depends on immobilization of the sample to the mounting surface. Optimally, immobilization should be minimally invasive to the sample such that metabolic processes and functional attributes are not compromised. By coating freshly cleaved mica surfaces with porcine (pig) gelatin, negatively charged bacteria can be immobilized on the surface and imaged in liquid by AFM. Immobilization of bacterial cells on gelatin-coated mica is most likely due to electrostatic interaction between the negatively charged bacteria and the positively charged gelatin. Several factors can interfere with bacterial immobilization, including chemical constituents of the liquid in which the bacteria are suspended, the incubation time of the bacteria on the gelatin coated mica, surface characteristics of the bacterial strain and the medium in which the bacteria are imaged. Overall, the use of gelatin-coated mica is found to be generally applicable for imaging microbial cells.Download video file.^{(62M, mov)}     

Probing specific molecular conformations with the scanning force microscope. Complexes of plasmid DNA and anti-Z-DNA antibodies.

An anti-Z-DNA IgG antibody was used to probe for the left-handed Z-DNA conformation of a d(CG)11 insert in a negatively supercoiled plasmid DNA (pAN022). The complexes were spread on mica in the presence of a quaternary ammonium detergent benzyldimethylalkylammonium chloride and imaged with a scanning force microscope (SFM). The high affinity anti-Z-DNA antibody was retained even after restriction endonuclease cleavage of the DNA. The two arms in the product molecules had unequal lengths in conformity with the known location of the Z-DNA forming insert. Most complexes exhibited one IgG per DNA molecule. The bound antibodies were up to approximately 35 nm in diameter and extended approximately 2 nm from the mica surface. They were generally in a lateral orientation relative to the DNA, in accordance with prior chemical modification experimental data indicating a bipedal mode of binding for an anti-Z-DNA IgG. However, the SFM images also suggest that the DNA bends to accommodate the two Fab combining regions of the antibody. This study demonstrates the utility of the SFM for investigating conformation-dependent molecular recognition.     

A 13C CP/MAS NMR spectroscopy and AFM study of the structure of Glucagel™, a gelling β-glucan from barley

The structure of Glucagel™, a mixed-linked (1→3), (1→4)-β-d-glucan extracted from barley, was examined using ¹³C CP/MAS NMR spectroscopy and atomic force microscopy (AFM). Results from ¹³C CP/MAS NMR spectroscopy showed that Glucagel™ contained regions with two distinct conformations. In some of the regions the β-glucan chains associated to form a unique conformation, the A-conformation, while in the other regions the β-glucan chains were in an amorphous conformation. Dilute solutions of Glucagel™ were prepared for imaging by dissolving Glucagel™ in water at 90 °C. If the dilute solution was immediately deposited onto mica and the surface dried, then no fine detail was seen in the AFM image. However, when dilute solutions of Glucagel™ were left for several days before being deposited onto the mica surface, individual fibres could be clearly imaged. These results suggested that in gels formed from Glucagel™, junction zones occur because of the interaction of two β-glucan chains in the A-conformation.