Biological nano-assemblies for ultrabright light sources

This section deals with the characterisation and use of available periodic biological structures to build frameworks for biological nano-assemblies for use in ultrabright X-ray sources. As first goal, we want to use the new techniques to investigate natural biological assemblies, in particular components of the cytoskeleton. Recent research has underscored the role of the cytoskeleton as an essentially dynamic multifunctional muscle. In-vitro reconstitution of passive and active subunits or “functional modules” of the cytoskeleton is now seen as a way of balancing the mutually conflicting demands for simplicity. This bottom-up strategy aimed at unravelling biological complexity from its physical basis, relies heavily on latest advances in technology, experimental design, and theoretical modelling. We plan to investigate the structure of bundles, such as occur in filopodia or microvilli. For this it will be necessary to build quasi-crystals of bundled short actin filaments cross-linked by fascin or plastin. By modifying salt concentrations, and adding, for example depletion forces as additional attractive potential we plan to explore the parameters under which bigger bundles and finally small quasi-crystals of bundles can be observed. By raising the actin concentration a nematic phase of bundles is expected, which again can be used as a quasi crystal. These will then be observed in an environmental chamber in cooperation with C.3.4. In a next step we plan to add molecular motors to the bundles, which at a later stage will allow time-dependent observation of the power-strokes of motors.

Another goal is to create nanocrystals (nucleation complexes) out of biological specimens and produce defined biological polymers for use as frameworks for nano-assemblies. Crystallisation involves in general two phases, nucleation and subsequent crystal growth. Since at present only crystals are of interest, nucleation assemblies have been mostly ignored and the nucleation process of biological specimens is largely unexplored. However, the upcoming FEL sources make it possible to obtain suitable data from nano crystalline material of a hundred molecules or so. It is therefore highly desirable to investigate how suitable nucleation products can be obtained. Of particular interest are affinity-tagged purified complexes, which can be produced on a regular basis but typically fail to produce large crystals. We will use model systems and dynamic light scattering to derive principles for obtaining nanocrystalline material. Fishing and freezing techniques and low-resolution analysis of the properties will be investigated by electron microscopy (C.3.4). In addition, we will pioneer the use of biological nano-assemblies such as the Rad51 DNA: protein filament and natural 2D crystalline biological molecules as a framework for engineered nano-assemblies.