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.     

Unconventional Atomic Structure of Graphene Sheets on Solid Substrates

The atomic structure of free‐standing graphene comprises flat hexagonal rings with a 2.5 Å period, which is conventionally considered the only atomic period and determines the unique properties of graphene. Here, an unexpected highly ordered orthorhombic structure of graphene is directly observed with a lattice constant of ≈5 Å, spontaneously formed on various substrates. First‐principles computations show that this unconventional structure can be attributed to the dipole between the graphene surface and substrates, which produces an interfacial electric field and induces atomic rearrangement on the graphene surface. Further, the formation of the orthorhombic structure can be controlled by an artificially generated interfacial electric field. Importantly, the 5 Å crystal can be manipulated and transformed in a continuous and reversible manner. Notably, the orthorhombic lattice can control the epitaxial self‐assembly of amyloids. The findings reveal new insights about the atomic structure of graphene, and open up new avenues to manipulate graphene lattices.     

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.     

In situ monitoring of single molecule binding reactions with time-lapse atomic force microscopy on functionalized DNA origami

Individual biomolecular binding events were recorded in situ by combining time-lapse atomic force microscopy and DNA origami. Single streptavidin molecules bound to specifically biotinyated DNA origami were simply counted as a function of time to obtain a direct measure of the binding rate.     

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.     

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.     

Inhibition of protein adsorption to muscovite mica by monovalent cations

Global analysis of fluorescence lifetime image microscopy (FLIM) data can be used to obtain an accurate fit of multi‐exponential fluorescence decays. In particular, it can be used to fit a bi‐exponential decay to single frequency FLIM data, which is not possible with conventional fitting techniques. Bi‐exponential fluorescence decay models can be used to analyse quantitatively single frequency FLIM data from samples that exhibit fluorescence resonance energy transfer (FRET). Global analysis algorithms simultaneously fit multiple measurements acquired under different experimental conditions to achieve higher accuracy. To demonstrate that bi‐exponential models can indeed be fitted to single frequency data, we derive an analytical solution for the special case of two measurements and use this solution to illustrate the properties of global analysis algorithms. We also derive a novel global analysis algorithm that is optimized for single frequency FLIM data, and demonstrate that it is superior to earlier algorithms in terms of computational requirements.     

Editorial

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 30~40°, 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.