Daniel M. Czajkowsky

Biological atomic force microscopy: what is achieved and what is needed

Biological atomic force microscopy (AFM) has become a rapidly developing interdisciplinary field of research in recent years. Not only has the technique, including instrumentation and specimen preparation methods, become increasingly sophisticated, but also its applications have encompassed a broad range of interesting subjects in biology. In this review, we present an extensive overview of the current status of biological AFM, including both the instrumentation and the application of AFM. In addition, we discuss the major problems that have yet to be fully resolved and present our analysis of the various factors involved. The published results so far clearly demonstrate the great potential of AFM in structural research and the ability of AFM to make unique contributions to our comprehension of various biological processes.

Vertical collapse of a cytolysin prepore moves its transmembrane β-hairpins to the membrane

Perfringolysin O (PFO) is a prototype of the large family of pore‐forming cholesterol‐dependent cytolysins (CDCs). A central enigma of the cytolytic mechanism of the CDCs is that their membrane‐spanning β‐hairpins (the transmembrane amphipathic β‐hairpins (TMHs)) appear to be ∼40 Å too far above the membrane surface to cross the bilayer and form the pore. We now present evidence, using atomic force microscopy (AFM), of a significant difference in the height by which the prepore and pore protrude from the membrane surface: 113±5 Å for the prepore but only 73±5 Å for the pore. Time‐lapse AFM micrographs show this change in height in real time. Moreover, the monomers in both complexes exhibit nearly identical surface features and these results in combination with those of spectrofluorimetric analyses indicate that the monomers remain in a perpendicular orientation to the bilayer plane during this transition. Therefore, the PFO undergoes a vertical collapse that brings its TMHs to the membrane surface so that they can extend across the bilayer to form the β‐barrel pore.

Fc-fusion proteins: new developments and future perspectives

Since the first description in 1989 of CD4‐Fc‐fusion antagonists that inhibit human immune deficiency virus entry into T cells, Fc‐fusion proteins have been intensely investigated for their effectiveness to curb a range of pathologies, with several notable recent successes coming to market. These promising outcomes have stimulated the development of novel approaches to improve their efficacy and safety, while also broadening their clinical remit to other uses such as vaccines and intravenous immunoglobulin therapy. This increased attention has also led to non‐clinical applications of Fc‐fusions, such as affinity reagents in microarray devices. Here we discuss recent results and more generally applicable strategies to improve Fc‐fusion proteins for each application, with particular attention to the newer, less charted areas.

High-resolution atomic-force microscopy of DNA: the pitch of the double helix

Using a cationic lipid bilayer, we show that DNA can be reliably adsorbed to the bilayer surface for atomic force microscopy (AFM) in aqueous buffers at high resolution. The measured width of the dsDNA is close to 2 nm, and a periodic modulation on dsDNA is reproducibly detected by the AFM. The measured period is 3.4 ± 0.4nm, in excellent agreement with the known pitch of the double helix. The right‐handedness of the double helix is directly discernible in high resolution AFM images. Thus, this approach can be readily applied to the study of DNA‐protein interactions, as well as sequence mapping at high resolution.

The human IgM pentamer is a mushroom-shaped molecule with a flexural bias

The textbook planar model of pentameric IgM, a potent activator of complement C1q, is based upon the crystallographic structure of IgG. Although widely accepted, key predictions of this model have not yet been directly confirmed, which is particularly important since IgG lacks a major Ig fold domain in its Fc region that is present in IgM. Here, we construct a homology-based structural model of the IgM pentamer using the recently obtained crystallographic structure of IgE Fc, which has this additional Ig domain, under the constraint that all of the cysteine residues known to form disulfide bridges both within each monomer and between monomers are bonded together. In contrast to the planar model, this model predicts a non-planar, mushroom-shaped complex, with the central portion formed by the C-terminal domains protruding out of the plane formed by the Fab domains. This unexpected conformation of IgM is, however, directly confirmed by cryo-atomic force microscopy of individual human IgM molecules. Further analysis of this model with free energy calculations of out-of-plane Fab domain rotations reveals a pronounced asymmetry favoring flexions toward the central protrusion. This bias, together with polyvalent attachment to cell surface antigen, would ensure that the IgM pentamer is oriented on the cell membrane with its C1q binding sites fully exposed to the solution, and thus provides a mechanistic explanation for the first steps of C1q activation by IgM.

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pyloristrains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Δ6–27)) in which this hydrophobic segment was deleted. VacA-(Δ6–27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Δ6–27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Δ6–27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Δ6–27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Δ6–27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Δ6–27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

VacA from Helicobacter pylori: a hexameric chloride channel

VacA is a unique protein toxin secreted by the human pathogen Helicobacter pylori. At a neutral pH, the cytotoxin self‐associates into predominantly dodecameric complexes. In this report, we show that at an acidic pH, VacA forms anion selective channels in planar phospholipid bilayers. Similar to several other chloride channels, the VacA channel exhibits a moderate selectivity for anions over cations (PCl:PNa=4.2:1), inhibition by the blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid and a permeability sequence, SCN−≫I−>Br−>Cl−>F, consistent with a ‘weak field strength’ binding site for the permeant anion. Single channel recordings reveal rapid transitions (486 s−1) between the closed state and a single open state of 24 pS (+60 mV, 1.5 M NaCl). Evaluation of the rate of increase in macroscopic current as well as atomic force microscopy suggest that this VacA channel is a hexamer, formed by the assembly of membrane‐bound monomers. Not only are these VacA channels likely to play an important role in the pathological activity of this toxin, but they may also serve as a model system to further investigate the mechanism of anion selectivity in general.

Measurement of the electron-phonon coupling factor dependence on film thickness and grain size in Au, Cr, and Al.

Femtosecond thermoreflectance data for thin films and bulk quantities of Au, Cr, and Al are compared with the parabolic two-step thermal diffusion model for the purpose of determining the electron-phonon coupling factor. The thin films were evaporated and sputtered onto different substrates to produce films that vary structurally. The measurement of the electron-phonon coupling factor is shown to be sensitive to grain size and film thickness. The thin-film thermoreflectance data are compared with that of the corresponding bulk material and to a theoretical model relating the coupling rate to the grain-boundary scattering and size effects on the mean free path of the relevant energy carrier.

Submolecular resolution of single macromolecules with atomic force microscopy

The intrinsically high signal‐to‐noise ratio of atomic force microscopy (AFM) permits structural determination of individual macromolecules to, at times, subnanometer resolution directly from unprocessed images, avoiding the conditions and possible consequences of averaging over an ensemble of molecules. In this article, we will review some of the most recent achievements in imaging single macromolecules with AFM.

High resolution surface structure of E. coli GroES oligomer by atomic force microscopy

Using atomic force microscopy (AFM) in aqueous solution, we show that the surface structure of the oligomeric GroES can be obtained up to 10 Å resolution. The seven subunits of the heptamer were well resolved without image averaging. The overall dimension of the GroES heptamer was 8.4±0.4 nm in diameter and 3.0±0.3 nm high. However, the AFM images further suggest that there is a central protrusion of 0.8±0.2 nm high and 4.5±0.4 nm in diameter on one side of GroES which displays a profound seven‐fold symmetry. It was found that GroEL could not bind to the adsorbed GroES in the presence of AMP‐PNP and Mg2+, suggesting that the side of GroES with the central protrusion faces away from the GroEL lumen, because only one side of GroES was observed under these conditions. Based on the results from both electron and atomic force microscopy, a surface model for the GroES is proposed.