Micro to macro scale simulation coupling for stray light analysis.

Stray light in an optical system is unwanted parasitic light that may degrade performance. It can originate from different sources and may lead to different problems in the optical system such as fogging, ghost images for imagers, or inaccurate measurements for time of flight applications. One of the root causes is the reflectivity of the sensor itself. In this paper we present a new optical simulation methodology to analyze the stray light contribution due to the sensor reflectivity by coupling electromagnetic simulation (to calculate the pixels' bidirectional reflectance distribution function, also named BRDF) and ray-tracing simulation (for stray light analysis of the camera module). With this simulation flow we have been able to reproduce qualitatively red ghost images observed on different sensors in our laboratory.

Collective osmotic shock in ordered materials.

Osmotic shock in a vesicle or cell is the stress build-up and subsequent rupture of the phospholipid membrane that occurs when a relatively high concentration of salt is unable to cross the membrane and instead an inflow of water alleviates the salt concentration gradient. This is a well-known failure mechanism for cells and vesicles (for example, hypotonic shock) and metal alloys (for example, hydrogen embrittlement). We propose the concept of collective osmotic shock, whereby a coordinated explosive fracture resulting from multiplexing the singular effects of osmotic shock at discrete sites within an ordered material results in regular bicontinuous structures. The concept is demonstrated here using self-assembled block copolymer micelles, yet it is applicable to organized heterogeneous materials where a minority component can be selectively degraded and solvated whilst ensconced in a matrix capable of plastic deformation. We discuss the application of these self-supported, perforated multilayer materials in photonics, nanofiltration and optoelectronics.

Alpha-helical peptide assemblies giving new function to designed structures.

The design of alpha-helical tectons for self-assembly is maturing as a science. We have now reached the point where many different coiled-coil topologies can be reliably produced and validated in synthetic systems and the field is now moving on towards more complex, discrete structures and applications. Similarly the design of infinite or fiber assemblies has also matured, with the creation fibers that have been modified or functionalized in a variety of ways. This chapter discusses the progress made in both of these areas as well as outlining the challenges still to come.

Imaging internal features of whole, unfixed bacteria.

Wet scanning-transmission electron microscopy (STEM) is a technique that allows high-resolution transmission imaging of biological samples in a hydrated state, with minimal sample preparation. However, it has barely been used for the study of bacterial cells. In this study, we present an analysis of the advantages and disadvantages of wet STEM compared with standard transmission electron microscopy (TEM). To investigate the potential applications of wet STEM, we studied the growth of polyhydroxyalkanoate and triacylglycerol carbon storage inclusions. These were easily visible inside cells, even in the early stages of accumulation. Although TEM produces higher resolution images, wet STEM is useful when preservation of the sample is important or when studying the relative sizes of different features, since samples do not need to be sectioned. Furthermore, under carefully selected conditions, it may be possible to maintain cell viability, enabling new types of experiments to be carried out. To our knowledge, internal features of bacterial cells have not been imaged previously by this technique.

The non-covalent decoration of self-assembling protein fibers.

The design of self-assembling fibers presents challenges in basic science, and has potential for developing materials for applications in areas such as tissue engineering. A contemporary issue in the field is the construction of multi-component, functionalized systems. Previously, we have developed peptide-based fibers, the SAF system, that comprises two complementary peptides, which affords considerable control over assembly and morphology. Here we present a straightforward route to functionalizing the SAFs with small molecules and, subsequently, other moieties. This is achieved via non-covalent recruitment of charged peptide tags, which offers advantages such as further control, reversibility, and future prospects for developing recombinant tags. We demonstrate the concept by appending fluorescent labels and biotin (and thence gold nanoparticles) to the peptides, and visualising the resulting decorated SAFs by light and electron microscopy. The peptide tags bind in the nm-mum range, and show specificity compared with control peptides, and for the SAFs over similar alpha-helix-based peptide fibers.

Assembly pathway of a designed alpha-helical protein fiber.

Interest in the design of peptide-based fibrous materials is growing because it opens possibilities to explore fundamental aspects of peptide self-assembly and to exploit the resulting structures--for example, as scaffolds for tissue engineering. Here we investigate the assembly pathway of self-assembling fibers, a rationally designed alpha-helical coiled-coil system comprising two peptides that assemble on mixing. The dimensions spanned by the peptides and final structures (nanometers to micrometers), and the timescale over which folding and assembly occur (seconds to hours), necessitate a multi-technique approach employing spectroscopy, analytical ultracentrifugation, electron and light microscopy, and protein design to produce a physical model. We show that fibers form via a nucleation and growth mechanism. The two peptides combine rapidly (in less than seconds) to form sticky ended, partly helical heterodimers. A lag phase follows, on the order of tens of minutes, and is concentration-dependent. The critical nucleus comprises six to eight partially folded dimers. Growth is then linear in dimers, and subsequent fiber growth occurs in hours through both elongation and thickening. At later times (several hours), fibers grow predominantly through elongation. This kinetic, biomolecular description of the folding-and-assembly process allows the self-assembling fiber system to be manipulated and controlled, which we demonstrate through seeding experiments to obtain different distributions of fiber lengths. This study and the resulting mechanism we propose provide a potential route to achieving temporal control of functional fibers with future applications in biotechnology and nanoscale science and technology.

Flow linear dichroism of some prototypical proteins.

Flow linear dichroism (LD) spectroscopy provides information on the orientation of molecules in solution and hence on the relative orientation of parts of molecules. Long molecules such as fibrous proteins can be aligned in Couette flow cells and characterized using LD. We have measured using Couette flow and calculated from first principles the LD of proteins representing prototypical secondary structure classes: a self-assembling fiber and tropomyosin (all-alpha-helical), FtsZ (an alphabeta protein), an amyloid fibril (beta-sheet), and collagen [poly(proline)II helices]. The combination of calculation and experiment allows elucidation of the protein orientation in the Couette flow and the orientation of chromophores within the protein fibers.

Efficient energy transfer within self-assembling peptide fibers: a route to light-harvesting nanomaterials.

Small peptides offer an attractive starting point for the development of self-assembling materials for a variety of purposes, since they are relatively simple to produce and can be tailored to provide an expansive range of chemical functionality. We have employed a short peptide that spontaneously self-assembles into a multimolecular fibrillar architecture to drive the coassembly of two independent luminescent moieties. We use fluorescence spectroscopy to demonstrate that the resulting complex performs a light-harvesting function.

MagicWand: a single, designed peptide that assembles to stable, ordered alpha-helical fibers.

We describe a straightforward single-peptide design that self-assembles into extended and thickened nano-to-mesoscale fibers of remarkable stability and order. The basic chassis of the design is the well-understood dimeric alpha-helical coiled-coil motif. As such, the peptide has a heptad sequence repeat, abcdefg , with isoleucine and leucine residues at the a and d sites to ensure dimerization. In addition, to direct staggered assembly of peptides and to foster fibrillogenesisthat is, as opposed to blunt-ended discrete speciesthe terminal quarters of the peptide are cationic and the central half anionic with lysine and glutamate, respectively, at core-flanking e and g positions. This +,-,-,+ arrangement gives the peptide its name, MagicWand (MW). As judged by circular dichroism (CD) spectra, MW assembles to alpha-helical structures in the sub-micromolar range and above. The thermal unfolding of MW is reversible with a melting temperature >70 degrees C at 100 muM peptide concentration. Negative-stain transmission electron microscopy (TEM) of MW assemblies reveals stiff, straight, fibrous rods that extended for tens of microns. Moreover, different stains highlight considerable order both perpendicular and parallel to the fiber long axis. The dimensions of these features are consistent with bundles of long, straight coiled alpha-helical coiled coils with their axes aligned parallel to the long axis of the fibers. The fiber thickening indicates inter-coiled-coil interactions. Mutagenesis of the outer surface of the peptide i.e., at the b and f positionscombined with stability and microscopy measurements, highlights the role of electrostatic and cation-pi interactions in driving fiber formation, stability and thickening. These findings are discussed in the context of the growing number of self-assembling peptide-based fibrous systems.

Synthetic biology through biomolecular design and engineering.

Synthetic biology is a rapidly growing field that has emerged in a global, multidisciplinary effort among biologists, chemists, engineers, physicists, and mathematicians. Broadly, the field has two complementary goals: To improve understanding of biological systems through mimicry and to produce bio-orthogonal systems with new functions. Here we review the area specifically with reference to the concept of synthetic biology space, that is, a hierarchy of components for, and approaches to generating new synthetic and functional systems to test, advance, and apply our understanding of biological systems. In keeping with this issue of Current Opinion in Structural Biology, we focus largely on the design and engineering of biomolecule-based components and systems.

Modification of fluorophore photophysics through peptide-driven self-assembly.

We describe the formation of self-assembling nanoscale fibrillar aggregates from a hybrid system comprising a short polypeptide conjugated to the fluorophore fluorene. The fibrils are typically unbranched, approximately 7 nm in diameter, and many microns in length. A range of techniques are used to demonstrate that the spectroscopic nature of the fluorophore is significantly altered in the fibrillar environment. Time-resolved fluorescence spectroscopy reveals changes in the guest fluorophore, consistent with energy migration and excimer formation within the fibrils. We thus demonstrate the use of self-assembling peptides to drive the assembly of a guest moiety, in which novel characteristics are observed as a consequence. We suggest that this method could be used to drive the assembly of a wide range of guests, offering the development of a variety of useful, smart nanomaterials that are able to self-assemble in a controllable and robust fashion.

Peptide and protein building blocks for synthetic biology: from programming biomolecules to self-organized biomolecular systems.

There are several approaches to creating synthetic-biological systems. Here, we describe a molecular-design approach. First, we lay out a possible synthetic-biology space, which we define with a plot of complexity of components versus divergence from nature. In this scheme, there are basic units, which range from natural amino acids to totally synthetic small molecules. These are linked together to form programmable tectons, for example, amphipathic alpha-helices. In turn, tectons can interact to give self-assembled units, which can combine and organize further to produce functional assemblies and systems. To illustrate one path through this vast landscape, we focus on protein engineering and design. We describe how, for certain protein-folding motifs, polypeptide chains can be instructed to fold. These folds can be combined to give structured complexes, and function can be incorporated through computational design. Finally, we describe how protein-based systems may be encapsulated to control and investigate their functions.

Possibilities for 'smart' materials exploiting the self-assembly of polypeptides into fibrils.

Programmed assembly and self-assembly of soft materials offers significant promise for the generation of new types of materials with useful properties. Through evolutionary processes occurring over billions of years, nature has produced numerous optimised building blocks for the controlled assembly of a wide range of complex architectures. Our challenge now is to imitate these naturally occurring processes for technological applications, either using biological molecules such as DNA and proteins, or macromolecular mimics that retain many of the important features of biological molecules while introducing new functionalities. We focus on a single example of biomolecular self-assembly-the self-assembly of polypeptides, including polypeptide mimics, into quasi-one-dimensional fibres-to provide a flavour of the utility of soft biological materials for construction purposes.