Biomedical subjects

D M Czajkowsky

Publications and source records attributed to D M Czajkowsky.

15 recordsLinked to original sources

PubMed2003

Inhibition of protein adsorption to muscovite mica by monovalent cations.

One of the most challenging steps in biological atomic force microscopy (AFM) is to find conditions under which the sample will adsorb to a substrate. Here we show that a common constituent of biological buffers, monovalent cations, can inhibit the adsorption of a number of different proteins onto one of the best substrates for biological AFM, muscovite mica. The potency series for different cations to prevent adsorption is the same for every protein, K+ > Na+ > Li+, and, in each case, this inhibition could be overcome by increasing the concentration of proteins. These results thus suggest that reducing the extent of this inhibition by using lower concentrations of salt, higher concentrations of proteins, or Li+ in place of K+ and Na+ may be generally useful procedures to maximize the amount of protein on mica.

Adsorption↗

PubMed2000

Atomic force microscopy in structural biology: from the subcellular to the submolecular.

Atomic force microscopy (AFM) is capable of generating images within ranges of resolution that are of particular interest in biology. Although atomic resolution may not be possible with biological samples, a great deal of information can still be obtained from images that provide structures at a slightly lower level of resolution. The submolecular resolution images of bacteriorhodopsin and the chaperonin GroES, which revealed, respectively, individual loops and beta-turns, confirmed and complemented other structural investigations, while the molecular-level features in images of membrane-bound VacA, a cytotoxin from Helicobacter pylori, immediately suggested the possibility, subsequently proven, of channel-forming ability. A series of images with macromolecular resolution directly provided details on the mechanisms by which RNA polymerase nonspecifically translocates along DNA, and images with subcellular resolving power of erythrocytic cellular membranes showed, with unambiguous clarity, linear arrays of molecular complexes. In this review, we will describe some of the most biologically relevant findings that have been obtained with AFM within ranges of resolution from the submolecular to the molecular, and from the macromolecular to the subcellular. Furthermore, we will describe some of the sample conditions and imaging environments that are likely important to achieve a particular level of resolution.

Bacterial Proteins↗

PubMed1999

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

Most Helicobacter pylori strains 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-(Delta6-27)) in which this hydrophobic segment was deleted. VacA-(Delta6-27) 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-(Delta6-27) 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-(Delta6-27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Delta6-27) 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-(Delta6-27) 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-(Delta6-27) 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.

Amino Acid Sequence↗

PubMed1999

Images of oligomeric Kv beta 2, a modulatory subunit of potassium channels.

The Shaker type voltage-gated potassium (K+) channel consists of four pore-forming Kv alpha subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kv beta subunits via formation of heteromultimeric Kv alpha-Kv beta complexes. Because each (Kv alpha)4 could recruit more than one Kv beta subunit and different Kv beta subunits could potentially interact, the stoichiometry of alpha-beta and beta-beta complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kv beta 2 subunit in Sf9 insect cells. The purified Kv beta 2, examined by atomic force and electron microscopy techniques, is found predominately as a square-shaped tetrameric complex with side dimensions of 100 x 100 A2 and height of 51 A. Thus, Kv beta 2 is capable of forming a tetramer in the absence of pore-forming alpha subunits. The center of the Kv beta 2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.

Amino Acid Sequence↗

PubMed1999

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 (P(Cl):P(Na) = 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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1999

The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH.

Pathogenic strains of Helicobacter pylori secrete a cytotoxin, VacA, that in the presence of weak bases, causes osmotic swelling of acidic intracellular compartments enriched in markers for late endosomes and lysosomes. The molecular mechanisms by which VacA causes this vacuolation remain largely unknown. At neutral pH, VacA is predominantly a water-soluble dodecamer formed by two apposing hexamers. In this report, we show by using atomic force microscopy that below pH approximately 5, VacA associates with anionic lipid bilayers to form hexameric membrane-associated complexes. We propose that water-soluble dodecameric VacA proteins disassemble at low pH and reassemble into membrane-spanning hexamers. The surface contour of the membrane-bound hexamer is strikingly similar to the outer surface of the soluble dodecamer, suggesting that the VacA surface in contact with the membrane is buried within the dodecamer before protonation. In addition, electrophysiological measurements indicate that, under the conditions determined by atomic force microscopy for membrane association, VacA forms pores across planar lipid bilayers. This low pH-triggered pore formation is likely a critical step in VacA activity.

Bacterial Proteins↗

PubMed1999

AFM tips: how sharp are they?

From both simple estimates and a 'blind' reconstruction based on cryo-AFM images of filamentous actin, we find that the radius of curvature at the apex of Si3N4 tips can be as small as 1 nm with a conical angle in the range 30 approximately 40 degrees, revealing a relatively high aspect ratio that is much greater than previously anticipated. Our results show that commercially available cantilevers are often sharp enough for routine high resolution imaging of biological materials, and suggest that factors other than an inherent blunt tip are probably responsible for frequent occurrences of poor resolution.

Actins↗

PubMed1998

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.

Membrane Proteins↗

PubMed1998

Staphylococcal alpha-hemolysin can form hexamers in phospholipid bilayers.

Atomic force microscopy (AFM) was used to study the structure of the staphylococcal alpha-hemolysin (alpha HL) oligomer formed in supported phospholipid bilayers. In contrast to the recent X-ray crystallographic demonstration of a heptameric stoichiometry for the oligomer formed in deoxycholate (DOC) micelles, the high-resolution unprocessed AFM images unequivocally revealed a hexamer in these phospholipid bilayers. Independent support of this hexameric stoichiometry was obtained from the measurements of the lattice constant in the AFM images and from gel electrophoresis. Therefore, alpha HL can form two different, energetically stable oligomers, which differ in at least stoichiometry but perhaps subunit structure as well. Furthermore, stable, incomplete oligomers were observed in the AFM images, which may be of relevance to the mechanism by which alpha HL damages the cell.

Bacterial Toxins↗

PubMed1998

Direct visualization of surface charge in aqueous solution.

Tapping mode atomic force microscopy operated in the phase shift mode is used to directly visualize the surface charge on biological macromolecules under solution. A simple theory is presented which is qualitatively consistent with experimental observations, although a more complicated theoretical analysis is required for a quantitative comparison.

Lipid Bilayers↗

PubMed1996

Gramicidin A aggregation in supported gel state phosphatidylcholine bilayers.

Using an atomic force microscope, supported bilayers of saturated phosphatidylcholine (in the gel state) containing various amounts of gramicidin A (gA) were imaged in aqueous solutions and at room temperature. gA clusters were directly observed for the first time under these conditions. It was found that, at a lower gA concentration, gA aggregated into domains, composed of small clusters along with a considerable amount of lipids. This basic aggregation unit, most likely a hexamer, remained the same for acyl chain lengths from 14 to 18 carbons. These small clusters were observed to form elongated aggregates (line type) but never into extended pure gA domains. When gA concentrations were increased, for bilayers with 16 carbons or less, gA aggregated into larger domains but the basic unit remained separated by lipid molecules. At about 5 mol % gA, a percolation-like transition occurred at which the line type aggregates were connected to each other. However, for bilayers with more than 16 carbons, multiple lamellar structures were formed at higher gA fractions and the top layer had a ripple-like surface morphology. The molecular mechanism for the formation of these peculiar structures remains to be elucidated.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

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 angstroms 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.

Bacterial Proteins↗

PubMed1995

Ripple phase in asymmetric unilamellar bilayers with saturated and unsaturated phospholipids.

In a solution of phosphate-buffered saline (PBS), unilamellar bilayers with saturated phosphatidylcholines in one leaflet and negatively charged, unsaturated phospholipids in the other leaflet were observed in the ripple phase at room temperature using atomic force microscopy (AFM). This is the first observation of the ripple phase in asymmetric bilayers. Sodium and phosphate, components of PBS, were found to be necessary for the formation of the ripple structure in the asymmetric bilayers composed of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), demonstrating a dependency for specific ions for this phase. These results indicate that the two leaflets of a bilayer are closely coupled to give rise to such a long range and complicated morphology.

Ions↗

PubMed1995

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 +/- 0.4 nm, 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.

DNA↗

PubMed1993

Proton transfer from Asp-96 to the bacteriorhodopsin Schiff base is caused by a decrease of the pKa of Asp-96 which follows a protein backbone conformational change.

In the bacteriorhodopsin photocycle the transported proton crosses the major part of the hydrophobic barrier during the M to N reaction; in this step the Schiff base near the middle of the protein is reprotonated from D96 located near the cytoplasmic surface. In the recombinant D212N protein at pH > 6, the Schiff base remains protonated throughout the photocycle [Needleman, Chang, Ni, Váró, Fornés, White, & Lanyi (1991) J. Biol. Chem. 266, 11478-11484]. Time-resolved difference spectra in the visible and infrared are described by the kinetic scheme BR-->K<==>L<==>N (-->N')-->BR. As evidenced by the large negative 1742-cm-1 band of the COOH group of the carboxylic acid, deprotonation of D96 in the N state takes place in spite of the absence of the unprotonated Schiff base acceptor group of the M intermediate. Instead of internal proton transfer to the Schiff base, the proton is released to the bulk, and can be detected with the indicator dye pyranine during the accumulation of N'. The D212N/D96N protein has a similar photocycle, but no proton is released. As in wild-type, deprotonation of D96 in the N state is accompanied by a protein backbone conformational change indicated by characteristic amide I and II bands. In D212N the residue D96 can thus deprotonate independent of the Schiff base, but perhaps dependent on the detected protein conformational change. This could occur through increased charge interaction between D96 and R227 and/or increased hydration near D96. We suggest that the proton transfer from D96 to the Schiff base in the wild-type photocycle is driven also by such a decrease in the pKa of D96.

Amino Acid Sequence↗