Peter C. M. Christianen

A virus-based single-enzyme nanoreactor

Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or physically anchored to a surface, which is often disadvantageous because it may lead to denaturation. In a natural environment, enzymes are present in a confined reaction space, which inspired us to develop a generic method to carry out single-enzyme experiments in the restricted spatial environment of a virus capsid. We report here the incorporation of individual horseradish peroxidase enzymes in the inner cavity of a virus, and describe single-molecule studies on their enzymatic behaviour. These show that the virus capsid is permeable for substrate and product and that this permeability can be altered by changing pH.

Macroscopic Hierarchical Surface Patterning of Porphyrin Trimers via Self-Assembly and Dewetting

The use of bottom-up approaches to construct patterned surfaces for technological applications is appealing, but to date is applicable to only relatively small areas (∼10 square micrometers). We constructed highly periodic patterns at macroscopic length scales, in the range of square millimeters, by combining self-assembly of disk-like porphyrin dyes with physical dewetting phenomena. The patterns consisted of equidistant 5-nanometer-wide lines spaced 0.5 to 1 micrometers apart, forming single porphyrin stacks containing millions of molecules, and were formed spontaneously upon drop-casting a solution of the molecules onto a mica surface. On glass, thicker lines are formed, which can be used to align liquid crystals in large domains of square millimeter size.

Photochemical Surface Patterning by the Thiol-Ene Reaction†

The immobilization of proteins on solid substrates while controlling the size and dimensions of the generated patterns is increasingly relevant in biotechnology. Site-specific immobilization and thus control over the orientation of proteins is particularly important because, as opposed to nonspecific adsorption, it generates homogeneous surface coverage and accessibility to the active site of the protein. Consequently, different types of bioorthogonal reactions have been developed to attach proteins site-specifically to surfaces and to control protein patterning. Herein, we report the photochemical coupling of olefins to thiols to generate a stable thioether bond for the covalent surface patterning of proteins and small molecules. This reaction has been applied previously in solution for carbohydrate and peptide coupling. The thiol-ene photoreaction proceeds at close to visible wavelengths (l = 365–405 nm) and in buffered aqueous solutions. As a result of its specificity for olefins, this photoreaction can be considered to be bioorthogonal, unlike other photochemical methods used previously for protein immobilization. To adopt the thiol-ene reaction for the immobilization of biomolecules, surfaces functionalized with thiols and biomolecules derivatized with olefins were prepared (Figure 1). Polyamidoamine (PAMAM) dendrimers were attached covalently to silicon oxide surfaces. An aminocaproic acid spacer was attached to the dendrimers to create distance from the surface. Cystamine was coupled to the spacer, and subsequent reduction of the disulfide yielded the desired thiolterminated surfaces. A liquid layer of terminal-olefinfunctionalized molecules dissolved in ethylene glycol was spread onto these wafers, which were then covered immediately with a photomask. Subsequent irradiation of the surfaces through the photomask led to patterning with adducts of covalently attached thioethers. To establish the method, we photochemically attached the biotin derivative 1 to a thiol-functionalized surface as described above (Figure 1). After the removal of unreacted biotin molecules, the surface was incubated with Cy5-labeled streptavidin (SAv) to produce a SAv-patterned surface. Fluorescence images of the resulting surface (Figure 1) demonstrated that lateral gradients and patterns with micrometer-sized features (5–100 mm) over areas of centimeters in width (Figure 1A) were readily accessible. Figure 1B,C and the fluorescence-intensity profile in Figure 1D show that the patterns have a well-defined shape and are homogeneous over large distances. When prolonged sonication (4 h) and stringent washing were carried our after irradiation, SAv patterns with similar fluorescence intensities were observed, whereas control experiments with biotin that lacked the olefin linker showed no distinctive SAv patterns. These results indicate that the covalent attachment of biotin to the surface occurs specifically through the proposed thiol-ene reaction and that the nonspecific adsorption of biotin is insignificant. Figure 1E shows that the amount of material immobilized can be modified by changing the irradiation time. The procedure reproducibly requires a short irradiation time of 60 s to yield sufficient surface coverage for fabricating dense SAv patterns. To obtain homogeneous fluorescence signals of the patterns, the starting concentration of the solution that is drop cast onto the surface is also important. When the solution of 1 was diluted (to 1 mm), the Cy5-fluorescence intensity decreased considerably. Further dilution (below 500 mm) resulted eventually in disrupted SAv patterns. The application of more concentrated solutions of 1 (> 20 mm) resulted in the saturation of the fluorescence intensity of the SAv patterns. This behavior corresponds well with the effects observed upon varying the irradiation time. Longer irradi[*] Dr. D. N sse, Dr. H. Schroeder, Dr. R. Wacker, Prof. Dr. C. M. Niemeyer Faculty of Chemistry Biological-Chemical Microstructuring Technical University of Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax: (+49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de

Ground-based facilities for simulation of microgravity: organism-specific recommendations for their use, and recommended terminology.

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organisms. However, research in the near-Earth orbit is severely constrained by the limited number of flight opportunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational research. The various microgravity simulators that are frequently used by gravitational biologists are based on different physical principles. This comparative study gives an overview of the most frequently used microgravity simulators and demonstrates their individual capacities and limitations. The range of applicability of the various ground-based microgravity simulators for biological specimens was carefully evaluated by using organisms that have been studied extensively under the conditions of real microgravity in space. In addition, current heterogeneous terminology is discussed critically, and recommendations are given for appropriate selection of adequate simulators and consistent use of nomenclature.

Selection of supramolecular chirality by application of rotational and magnetic forces

Many essential biological molecules exist only in one of two possible mirror-image structures, either because they possess a chiral unit or through their structure (helices, for example, are intrinsically chiral), but so far the origin of this homochirality has not been unraveled. Here we demonstrate that the handedness of helical supramolecular aggregates formed by achiral molecules can be directed by applying rotational, gravitational and orienting forces during the self-assembly process. In this system, supramolecular chirality is determined by the relative directions of rotation and magnetically tuned effective gravity, but the magnetic orientation of the aggregates is also essential. Applying these external forces only during the nucleation step of the aggregation is sufficient to achieve chiral selection. This result shows that an almost instantaneous chiral perturbation can be transferred and amplified in growing supramolecular self-assemblies, and provides evidence that a falsely chiral influence is able to induce absolute enantioselection.

Complete Chiral Symmetry Breaking of an Amino Acid Derivative Directed by Circularly Polarized Light

Circularly polarized light (CPL) emitted from star-forming regions is an attractive candidate as a cause of single chirality in nature. It has remained difficult, however, to translate the relatively small chemical effects observed on irradiation of molecular systems with CPL into high enantiomeric excesses. Here we demonstrate that irradiation of a racemic amino acid derivative with CPL leads to a small amount of chiral induction that can be amplified readily to give an enantiopure solid phase. A racemate composed of equal amounts of left- and right-handed crystals in contact with the irradiated solution is converted completely into crystals of single-handedness through abrasive grinding when racemization is effected in the solution. The rotation sense of the CPL fully determines the handedness of the final solid state. These findings illustrate the potential effectiveness of CPL in the control of molecular asymmetry, which is relevant for the origin of the single chirality inherent to many biological molecules.

Oligo(p-phenylenevinylene) peptide conjugates: Synthesis and self-assembly in solution and at the solid-liquid interface

Two oligo(p-phenylenevinylene)-peptide hybrid amphiphiles have been synthesized using solid- and liquid-phase strategies. The amphiliphiles are composed of a pi-conjugated oligo(p-phenylenevinylene) trimer (OPV) which is coupled at either a glycinyl-alanyl-glycinyl-alanyl-glycine (GAGAG) silk-inspired beta-sheet or a glycinyl-alanyl-asparagyl-prolyl-asparagy-alanyl-alanyl-glycine (GANPNAAG) beta-turn forming oligopeptide sequence. The solid-phase strategy enables one to use longer peptides if strong acidic conditions are avoided, whereas the solution-phase coupling gives better yields. The study of the two-dimensional (2D) self-assembly of OPV-GAGAG by scanning tunneling microscopy (STM) at the submolecular level demonstrated the formation of bilayers in which the molecules are lying antiparallel in a beta-sheet conformation. In the case of OPV-GANPNAAG self-assembled monolayers could not be observed. Absorption, fluorescence, and circular dichroism studies showed that OPV-GAGAG and OPV-GANPNAAG are aggregated in a variety of organic solvents. In water cryogenic temperature transmission electron microscopy (cryo-TEM), atomic force microscopy (AFM), light scattering, and optical studies reveal that self-assembled nanofibers are formed in which the helical organization of the OPV segments is dictated by the peptide sequence.

A virus-based biocatalyst

Virus particles are probably the most precisely defined nanometre-sized objects that can be formed by protein self-assembly. Although their natural function is the storage and transport of genetic material, they have more recently been applied as scaffolds for mineralization and as containers for the encapsulation of inorganic compounds. The reproductive power of viruses has been used to develop versatile analytical methods, such as phage display, for the selection and identification of (bio)active compounds. To date, the combined use of self-assembly and reproduction has not been used for the construction of catalytic systems. Here we describe a self-assembled system based on a plant virus that has its coat protein genetically modified to provide it with a lipase enzyme. Using single-object and bulk catalytic studies, we prove that the virus-anchored lipase molecules are catalytically active. This anchored biocatalyst, unlike man-made supported catalysts, has the capability to reproduce itself in vivo, generating many independent catalytically active copies.

Dilution-induced self-assembly of porphyrin aggregates: A consequence of coupled equilibria

The self-assembly of organic molecules has attracted sub-stantialinterest asa bottom-upapproachto createnano-sizedobjects. Their properties depend strongly on the design,arrangement, and number of molecules in the aggregate. Forsupramolecular polymers, the monomers are entirely heldtogether by non-covalent interactions; these interactions aretypically weak, reversible, and highly sensitive to variablessuch as temperature, concentration, and solvent polarity.

Influence of bismuth incorporation on the valence and conduction band edges of GaAs1−xBix

We investigate the electronic properties of GaAs1−xBix by photoluminescence at variable temperature (T=10–430K) and high magnetic field (B=0–30T). In GaAs0.981Bi0.019, localized state contribution to PL is dominant up to 150K. At T=180K the diamagnetic shift of the free-exciton states reveals a sizable increase in the carrier effective mass with respect to GaAs. Such an increase cannot be accounted for by an enhanced localized character of the valence band states, solely. Instead, it suggests that also the Bloch states of the conduction band are heavily affected by the presence of bismuth atoms.