Biochemical characterization of the Helicobacter pylori bactofilin-homolog HP1542.

The human pathogen Helicobacter pylori is known for its colonization of the upper digestive system, where it escapes the harsh acidic environment by hiding in the mucus layer. One factor promoting this colonization is the helical cell shape of H. pylori. Among shape determining proteins are cytoskeletal elements like the recently discovered bactofilins. Bactofilins constitute a widespread family of polymer-forming bacterial proteins whose biology is still poorly investigated. Here we describe the first biochemical analysis of the bactofilin HP1542 of H. pylori reference strain 26695. Purified HP1542 forms sheet-like 2D crystalline assemblies, which clearly depend on a natively structured C-terminus. Polymerization properties and protein stability were investigated. Additionally, we also could demarcate HP1542 from amyloid proteins that share similarities with the bactofilin DUF domain. By using zonal centrifugation of total H. pylori cell lysates and immunfluorescence analysis we revealed peripheral membrane association of HP1542 mostly pronounced near mid-cell. Interestingly our results indicate that H. pylori bactofilin does not contribute to cell wall stability. This study might act as a starting point for biophysical studies of the H. pylori bactofilin biology as well as for the investigation of bactofilin cell physiology in this organism. Importantly, this study is the first biochemical analysis of a bactofilin in a human pathogen.

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.

Bioinspired nanoencapsulation of purple membranes.

Embedding biological compounds, e.g. enzymes or whole cells, in solid host material seems to be a promising approach to widen their field of application far beyond the limits of natural conditions. In fields such as medicine and biotechnology, there is great interest in new methods to produce these types of composite materials in the form of micro- or nanosized particles. Such methods should be applicable to large amounts of substance. Inspired by one of nature's remarkable features-its ability to combine (bio)organic and inorganic components at the nanoscale-we developed a generic silica encapsulation method for biomolecules based on the concepts of polyelectrolyte layer adsorption followed by templated silica mineralization similar to biomineralization in diatoms. Application of this method to the model substance purple membrane (PM) resulted in a defined hybrid material with a nanoscale protective encapsulating silica shell providing a high barrier for the diffusion of low molecular weight molecules.

Oriented purple membrane monolayers covalently attached to gold by multiple thiole linkages analyzed by single molecule force spectroscopy.

Highly oriented monolayers of bacteriorhodopsin (BR) in purple membrane (PM) form are obtained by the reaction of BR-Q3C, where a cysteine was introduced into the N-terminal region, with a gold surface. Single molecule force spectroscopy was used to show that about 50% of the BRs are covalently bound to the surface. The linkage between the cysteine and the gold causes an additional characteristic peak in the force-distance curves to appear. Because several thousand cysteine-gold bonds exist between each PM patch and the surface, the PM is irreversibly bound. Such oriented PM monolayers may serve as an interface between metal surfaces and biomaterials, which may be linked to the PM surface chemically. Photoelectric applications of BR will benefit from the high degree of orientation obtained by this method.

Morphology of dry solid-supported protein monolayers dependent on the substrate and protein surface properties.

The morphologies of dry MrgA protein monolayers on different solid substrates prepared by a three-step procedure (adsorption from an incubation solution, rinsing to remove excess salt and protein, and drying) were investigated using atomic force microscopy. MrgA is a dodecameric iron-storage protein which can form hexagonal, two-dimensional (2D) crystalline monolayers on hydrophilic surfaces at low supersaturation. The formation of such two-dimensional crystals is heavily dependent on the pH and the salinity of the incubation solution as well as on the surface properties. Correlation of surface coverage with substrate charge, ionic strength, and pH indicates the dominance of electrostatic effects in adsorption, with the balance shifting between intermolecular repulsion and protein-substrate attraction. Close to the isoelectric point (pI) of MrgA, adsorption to the surface and the formation of 2D crystals are favored. By preparation of self-assembled monolayers of thiols with different end groups on template-stripped gold, the surface properties can be varied easily from high to very low protein affinity. The resulting patterns of the crystalline protein structures are novel and could be a starting point for further scientific study, e.g., solid-supported cocrystallization with DNA, and indeed developments with technological applications, such as mesostructured deposition of MrgA-caged nanoparticles.

Simultaneous removal of thiolated membrane proteins resulting in nanostructured lipid layers.

Self-organization of membrane-embedded peptides and proteins causes the formation of lipid mesostructures in the membranes. One example is purple membranes (PM), which consist of lipids and bacteriorhodopsin (BR) as the only protein component. The BRs form a hexagonal crystalline lattice. A complementary structure is formed by the lipids. Employing BR and PM as an example, we report a method where major parts of the mesoscopic self-assembled protein structures can be extracted from the lipid bilayer membrane. A complementary lipid nanostructure remains on the substrate. To remove such a large number of thiolated proteins simultaneously by applying a mechanical force, they are first reacted at physiological conditions with gold nanoparticles, and then a thin gold film is sputtered onto them that fuses with the gold nanoparticles forming a uniform layer, which finally can be lifted off. In this step, all of the previously gold-labeled proteins are pulled out of the membrane simultaneously. A stable lipid nanostructure is obtained on the mica substrate. Its stability is due to either binding of the lipids to the substrate through ionic bonds or to enough residual proteins to stabilize the lipid nanostructure against reorganization. This method may be applied easily and efficiently wherever thiolated proteins or peptides are employed as self-assembling and structure-inducing units in lipid membranes.

Long-chain fatty acids increase basal metabolism and depolarize mitochondria in cardiac muscle cells.

The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential (DeltaPsi) of intact guinea pig cardiac muscle were investigated at 37 degrees C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and DeltaPsi was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl-beta-cyclodextrin was used as fatty acid carrier. Application of 400 microM oleate or linoleate increased resting heat rate by approximately 30% and approximately 25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40-80 microM oleate nor 40 microM linoleate affected DeltaPsi. At a higher concentration (400 microM) both oleate and linoleate increased TMRE fluorescence by approximately 20% of maximum, obtained using 2,4-dinitrophenol (100 microM), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease DeltaPsi in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium.