Biomedical subjects

D M Czajkowsky

Publications and source records attributed to D M Czajkowsky.

7 recordsLinked to original sources

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↗

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↗