Cross-examining Polyurethane Nanodomain Formation and Internal Structure.

Structural and morphological interplay between hard and soft phases determine the bulk properties of thermoplastic polyurethanes. Commonly employed techniques rely on different physical or chemical phenomena for characterizing the organization of domains, but detailed structural information can be difficult to derive. Here, total scattering pair distribution function (PDF) analysis is used to determine atomic-scale insights into the connectivity and molecular ordering and compared to the domain size and morphological characteristics measured by AFM, TEM, SAXS, WAXS, and solid-state NMR 1H-1H spin-diffusion. In particular, density distribution functions are highlighted as a means to bridging the gap from the domain morphology to intradomain structural ordering. High real-space resolution PDFs are shown to provide a sensitive fingerprint for indexing aromatic, aliphatic, and polymerization-induced bonding characteristics, as well as the hard phase structure, and indicate that hard phases coexist in both ordered and disordered states.

Measuring interactions between yeast cells and a micro-sized air bubble via atomic force microscopy.

A method for the determination of interactions between yeast cells and air bubbles using the atomic force microscope was developed, in which a bubble acts as probe on immobilised living cells. The experimental setup and influencing parameters like bubble size, dwell time and maximum contact force on force-distance curves and maximum adhesion forces are explained. Also, interactions between bubble and yeast cells under variation of pH, ethanol concentration, salt concentration, ionic strength and influence of storage time in Yeast Malt Broth and phosphate buffered saline are investigated and discussed.

Structural changes in bacteriorhodopsin caused by two-photon-induced photobleaching.

Bacteriorhodopsin (BR) is the key protein of the halobacterial photosynthetic system. BR assembles into two-dimensional crystalline patches, the so-called purple membranes (PM), and acts as a light-driven proton pump converting light energy into the chemical energy of a proton gradient over the cell membrane. The two-photon absorption (TPA) of BR is so far not fully understood. Astonishingly high TPA cross sections have been reported, but the molecular mechanisms have not been elucidated. In this work, we address structural changes in BR and PM upon TPA, investigating its TPA photochemistry by spectroscopy, small-angle X-ray scattering, as well as electron and atomic force microscopy. We observe that TPA of BR leads to formation of an UV-absorbing N-retinyl-bacterioopsin state, which is accompanied by the loss of crystalline order in PM. FTIR and CD spectroscopy confirm that BR trimers as well as the secondary structure of the BR molecules are preserved. We demonstrate that excitation by TPA results in the photochemical reduction of the retinal Schiff base, which in turn causes a permanent asymmetric shape change of BR, similar to the one transiently observed during the photocycle-related opening and closing of the cytoplasmic proton half channel. This shape change causes PM sheets to merely roll up toward the extracellular side and causes the loss of crystallinity of PM. We present a model for the TPA photoresponse of BR, which also explains the irreversibility of the process in terms of a photochemical reduction of the Schiff base.

A new class of purple membrane variants for the construction of highly oriented membrane assemblies on the basis of noncovalent interactions.

Purple membranes (PM) from Halobacterium salinarum have been discussed for several technical applications. These ideas started just several years after its discovery. The biological function of bacteriorhodopsin (BR), the only protein in PM, is the light-driven proton translocation across the membrane thereby converting light energy into chemical energy. The astonishing physicochemical robustness of this molecular assembly and the ease of its isolation triggered ideas for technical uses. All basic molecular functions of BR, that is, photochromism, photoelectrism, and proton pumping, are key elements for technical applications like optical data processing and data storage, ultrafast light detection and processing, and direct utilization of sunlight in adenosine 5'-triphospate (ATP) generation or seawater desalination. In spite of the efforts of several research groups worldwide, which confirmed the proof-of-principle for all these potential applications, only the photochromism-based applications have reached a technical level. The physical reason for this is that no fixation or orientation of the PMs is required. The situation is quite different for photoelectrism and proton pumping where the macroscopic orientation of PMs is a prerequisite. For proton pumping, in addition, the formation of artificial membranes which prevent passive proton leakage is necessary. In this manuscript, we describe a new class of PM variants with oppositely charged membrane sides which enable an almost 100% orientation on a surface, which is the key element for photoelectric applications of BR. As an example, the mutated BR, BR-E234R7, was prepared and analyzed. A nearly 100% self-orientation on mica was obtained.

Photochemistry of coumarin-functionalized SiO2 nanoparticles.

We describe the synthesis and photochemistry of coumarin-functionalized silica nanoparticles, which were prepared utilizing 7-[3-(triethoxysilyl)propanyloxy]coumarin (TPC) to attach coumarin as a photoactive group to the silica nanoparticle surface. The nanoparticle size and morphology were investigated by scanning electron microscopy, atomic force microscopy, and dynamic light scattering. The diameter of the spherical nanoparticles was determined by all three methods to be about 40 nm. The surface functionalization was characterized in the bulk by ζ-potential measurements and on the single-nanoparticle level by electrostatic force microscopy, where the difference in surface potential between TPC-modified and unmodified silica nanoparticles is measured. The degree of surface functionalization was determined by thermogravimetric analysis (TGA), and a theoretical limit of about 23,000 coumarin entities per nanoparticle was calculated. The photochemistry, and its reversibility, of the nanoparticle-attached coumarin entities was found to be quite different from the coumarin photochemistry in solution or on flat surfaces. Photodimerization with light of 355 nm and photocleavage with light of 254, 266, and 280 nm were analyzed by absorption and fluorescence spectroscopy. Following several cycles of photodimerization and photocleavage showed that the absorption change at 320 nm decreases from cycle to cycle. The coumarin layer on the nanoparticles was proven to be unchanged by TGA. The apparent loss of absorption change is due to the formation of interlinked nanoparticles during the dimerization-cleavage cycles. Because the coumarin groups on the inside of the obtained nanoparticle clusters are inaccessible to light, the amount of "uncleavable" dicoumarins increases, thus lowering the obtainable absorption change from cycle to cycle.

Crystallinity of purple membranes comprising the chloride-pumping bacteriorhodopsin variant D85T and its modulation by pH and salinity.

Self-assembly of membrane proteins inside the cell membrane critically depends on specific protein-protein and protein-lipid interactions. Purple membranes (PMs) from Halobacterium salinarum comprise wild-type bacteriorhodopsin (BR) and lipids only and form a 2-D crystalline lattice of P3 symmetry in the cell membrane. It is known that removal of the retinylidene residue as well as the exchange of selected amino acids lead to a loss of crystallinity. In PMs comprising the BR variant D85T, we have observed a tunable tendency to form crystalline domains, which depends on pH-value and chloride ion concentration. BR-D85T resembles the function of the chloride pump halorhodopsin. The protonation state of amino acid residues within the binding pocket and chloride binding in the vicinity of the protonated retinal Schiff base affect the overall shape of BR-D85T molecules in the membrane, thereby changing their interactions and subsequently their tendency to form crystalline areas. The combination of small-angle X-ray scattering, atomic force microscopy, and freeze-fracture electron microscopy enables us to analyze the transitions statistically as well as on the single membrane level. PM-D85T is a model system to study membrane protein association upon substrate binding in a native environment. Furthermore, the ability to reversibly modulate the crystallinity of PMs probably will be useful for the preparation of larger artificial crystalline arrays of BR and its variants.

Dynamics of bacteriorhodopsin in solid-supported purple membranes studied with tapping-mode atomic force microscopy.

Purple membrane (PM) from Halobacterium salinarum, which comprises bacteriorhodopsin (BR) and lipids only, has been employed by many groups as a model system to study the structure and dynamics of membrane proteins. Although the conformational dynamics of BR within PM has been extensively analyzed with subnanometer resolution by means of diffraction experiments and spectroscopic methods, as well, structural studies of dynamical transitions within single PMs are rare. In this work, we show that tapping-mode atomic force microscopy (TM-AFM) is ideally suited to study dynamical transitions within solid-supported PMs at the nanoscale. Time-dependent AFM analysis of solid-supported PMs shows that redistribution processes take place between a crystalline core region, featuring a height of approximately 5 nm, and a highly mobile rim region (approximately 4 nm in height). Furthermore, we discuss the influence of temperature and substrate on the equilibrium. The experiments are complemented by electrostatic force microscopy (EFM) of PM on mica. Beyond their importance for many physiological processes, dynamical transitions in biological membranes, as observed in this work, are of critical importance for all methods that make use of solid-supported membrane assemblies, either analytical tools or applications.