Developement of novel design concepts
Most mechanical analysis methods at the single molecule level only work with in vitro assays. A future challenge will be to report mechanical processes and measure forces on the single molecule level within living cells. Based on recent mechanical studies with GFP (Rief) We plan to develop recombinant force-sensors that can be integrated into force-bearing structures like the cytoskeleton or the mitotic spindle to report on the acting mechanical forces on the single molecule level (Rief, Gaub, Bausch). The optical readout of forces acting between selected molecular assemblies will become possible at the level of individual molecules within live cells. Beyond in vivo force sensing, such force sensors, when attached with one end to a surface could also be used to report hydrodynamic flow velocities close to surfaces in microfluidic devices (Wixforth) with interesting links to the research in area F.
Multi-scale theoretical modeling
To study the effect of mechanical load on nanosystems it will be important to link different simulation techniques in order to account for the broad range of time- and length-scales involved. Using first-principles Molecular-Dynamics in the Car-Parrinello scheme, highly complex mechanisms involving many internal coordinates can be resolved on an atomic level (Frank). Such simulations yield detailed information about the elementary steps involved in the reaction sequences and thus allow to understand complex chemistry on a physical basis. Combining quantum chemical ab-initio methods with Monte-Carlo methods, the elasticity of single molecules can be understood quantitatively and included in large-scale simulations by appropriately chosen force-field parameterization (Netz). Hybrid methods combining Finite-Element and Monte-Carlo techniques (Frey) may finally be used to study stress and strain fields in macromolecular assemblies such as the protein materials envisaged as force sensors. Single molecule experiments involving mechanical and/or electrical manipulations are subjected to non-equilibrium fluctuations. Theoretical studies (Hänggi) will explore the validity of fluctuation theorems which principally relate equilibrium features like thermodynamic potential differences to tailored non-equilibrium measures.
Natural molecular machines and assembly lines
Many cellular functions are based on the complex interplay of nanometer sized machines that convert chemical energy into mechanical work with efficiencies reaching almost 100%. To unravel the fundamental underlying principles of these machines, as well as to understand how small adaptations lead to a number of specialized functions we will combine efforts from the fields of biophysics, physical chemistry and structural biology. In particular, we aim at deciphering of the inner workings of two important and outstanding nanomachines: RNA Polymerases and DNA Polymerases. The Cramer laboratory has extensive experience in the structural biology of RNA polymerase II, which transcribes the genetic information in all plant and animals cells. DNA polymerases, the central enzymes in replication, are currently being investigated in the Carell laboratory. Both RNA Polymerases and DNA Polymerases can operate with a surprisingly high fidelity that is achieved through complex recognition and error correction schemes. Beyond biochemical and structural investigations, single molecule methods, which have been developed and applied to multiple biological systems in the Bräuchle and Michaelis groups, enable us to follow these mechanisms directly with high spatial and time resolution. We will investigate how polymerases can overcome obstacles such as proteins bound to the DNA or external hindering forces by manipulating with single molecule force spectroscopy methods (Bräuchle, Michaelis). The combination with theoretical modeling of the corresponding functional biological modules (Frey, Gerland) will provide important information about the physical mechanisms of these high fidelity molecular machines and possibly be a first step towards the design of novel nano-machines.
Artificial molecular machines and assembly lines
Going a step further developing concepts to artificially assemble molecular machines may be envisioned. The Gaub group plans to combine molecular recognition and local self-organisation of biological macromolecules with the excellent position control of atomic force techniques to assemble nanoscale building blocks which are ultimately to be brought in some functional relationship. Such experiments will provide the scientific foundation for a technology that allows directed assembly of nanoscale functional units in fluid environments.
Structure-function relation of biopolymers
Single molecule methods allow unprecedented views of the conformations and non-equilibrium dynamics of biopolymers such as RNA, DNA, cytoskeletal filaments and a broad class of proteins. Single molecule force spectroscopy (Gaub, Rief) complemented by quantum-mechanical ab-initio calculations (Netz) have given detailed insights into the molecular origin of biopolymer elasticity. Fluorescence methods (Bausch) combined with statistical mechanics calculations have resulted in a comprehensive understanding of conformations of cytoskeletal filaments (Frey). We plan to further exploit this established and successful synergy between experimental and theoretical groups to unravel the biophysical properties of biopolymers and macromolecular assemblies of nano-structured networks, where molecular structure at the nanoscale is intimately connected with function on a mesoscale.
The great variability of repetitive DNA sequences allows the discrimination of DNA traces by fingerprinting, but is also related to diseases such as fragile X and Huntington's. The expansion of these sequences during replication relies on the dynamics of structural defects, which will be studied by monitoring the response of repetitive DNA to external forces (Gaub) and comparing the results with signatures of the defect dynamics calculated by the Gerland group. Beyond this particular question the physics of the multi-functional biopolymer DNA will be explored to understand the interplay between sequence specificity, nano-scale confinement and thermal fluctuations (Frey, Gerland, Gaub).
The von Willebrand factor (VWF), an important blood coagulation protein, tends to increase its potential to bind blood platelets under increased shear rates. The “force-structure-function relationship” of the VWF will be studied by the Wixforth group using a surface acoustic wave based microfluidic device designed in close cooperation with Rädler and Simmel (areas F and G), optical tweezers (Gaub, Rief) and theoretical simulations (Netz, Frey). Comparing the results to the mutant form of VWF, we plan to cover the whole range from the macroscopic appearance of VWF diseases down to the molecular origin of its unusual behaviour in cell adhesion under hydrodynamic flow.
Multi-scale theoretical modeling
Upon combining the knowledge gained from single-molecule and force-sensor studies (Rief, Gaub) with fluorescence-imaging and micro-rheology (Bausch) we aim at achieving a fundamental physical understanding of the intimate connection between individual actin binding proteins (ABP) physico-chemical properties and the rheology of reconstituted in vitro networks and ultimately also in vivo systems which are central to understanding basic cellular functions such as locomotion, phagocytosis, and cytokinesis. A hierarchical, multi-scale modeling approach combining finite element with Monte Carlo simulations (Frey) will be adopted in which both detailed computational polymer network models that take into account the specific network architecture and the nano-mechanical properties of the ABPs (Rief) will be developed and validated experimentally (Bausch).