|
|
|
We will employ AFM-based single molecule force spectroscopy in the nano-apertures of zero mode waveguide to activate molecular force sensors while we simultaneously record via fluorescence the binding of labeled ATP and substrates to the kinases. Titin Kinase will be compared to Myosin Light Chain Kinases and Focal Adhesion Kinase. Novel molecular constructs will be developed to allow for a stable and reliable handling of the kinases.
|
|
Single molecule mechanical methods offer unique possibilities for studying protein folding processes on fast and slow timescales. In this project, we will exploit those possibilities and study important events in protein folding. In the first part of the project, we will study the folding the ultra-fast folding protein villin headpiece under load. Moreover, we will try to measure directly the transition path time of a folding protein. In the second part of the project, we will investigate how proline isomerization, a key determinant for protein folding kinetics couples to mechanical forces. We will study on-line with a single molecule how the isomerization kinetics is altered by prolyl cis-trans isomerases.
|
|
We will investigate mechanical aspects of eukaryotic transcription by studying transcription elongation of single polymerases using high-resolution optical tweezers. Using either assisting or opposing forces we will compare the behavior of both RNA polymerase I as well as RNA polymerase II. By performing experiments in presence or absence of different transcription factors such as TFIIS and TFIIF, whose function is known biochemically we will gain insight into transcription regulation from a mechanical perspective. Thus, the experiments will further our understanding of eukaryotic transcription, a highly regulated, mechanical process.
|
|
The molecular chaperone Hsp90 is a dimeric ATP-driven molecular machine. Large conformational changes result in the reorientation of domains which are connected by flexible linkers. We will use an integrated approach comprising cutting edge single molecule mechanical and fluorescent methods as well as in vivo experiments to delineating the forces that are required to maintain specific conformations and domain orientations. In addition, we will address the question how the mechanics of Hsp90 are controlled by cochaperones and client proteins.
|
|
In a first part, we will determine the adhesion of various proteins that interact with lipid bilayers. To this aim, the proteins will be immobilized via a linker to an AFM-tip, pushed into the supported lipid bilayer and pulled off. Varying the lipid composition, temperature and buffers will allow understanding and controlling protein adhesion properties.
In a second part, we will measure the adhesion of selected biofilm polymers and lipids onto various solid substrates and to already formed biofilms. The goal is to understand which components of a biofilm generate their exceptional adhesion and self-healing properties.
|
|
Understanding the structure and dynamics of unfolded and partially folded states of proteins is essential for the elucidation of the free energy landscape for protein folding reactions. We will use a combination of fluorescence resonance energy transfer (FRET) and triplet-triplet energy transfer (TTET) experiments to determine dimensions and intrachain diffusion constants of model polypeptide chains, unfolded proteins and folding intermediates. These experiments will yield effective frictional coefficients for intrachain dynamics which will allow us to calculate frictional forces arising from chain motions in mechanical folding/unfolding experiments.
|
|
Because of hydration and the high degree of entanglement during protein folding, assembly, or translocation, friction forces govern the response of proteins to external perturbations. The aim of this project is the theoretical study of intra-chain and solvent-induced friction in proteins for a detailed, microscopic understanding of biomolecular mechanics. In close collaboration with the experiments in A2, A6, and A8, the dynamics and kinetic response of protein fragments will be studied using atomistic and coarse-grained computer simulations, leading finally to a reduced Fokker-Planck-type description of diffusion in a schematic, rough energy landscape.
|
|
SFB 863 TPA8 builds on the recent development of an artificial ion channel entirely constructed from DNA. Within this project, such DNA-based membrane channels will be custom-designed in shape, size and chemical functionality, which may be utilized for the improvement of DNA and RNA translocation studies. In addition, membrane-embedded DNA structures will be also utilized as nanomechanical components, e.g., as artificial molecular rotors.
|
|
Protein function is often tied to the dynamic interconversion between distinct molecular structures. In many cases, forces play a direct role in influencing the conformational switching of a protein. Studying the conformational dynamics of folded protein molecules under force has remained experimentally challenging owing to limitations of state-of-the-art instrumentation. In this project we propose to employ molecular self-assembly with DNA origami to develop a molecular toolbox that enables studying the conformational dynamics of single folded protein molecules under small force loads with dual-beam optical traps as well as with single-molecule FRET microscopy. We plan to apply our toolbox to investigate how force as a ligand mimic can control the conformational switching of adenylate kinase and calmodulin.
|
|
Protein molecules can undergo large scale motions such as folding/unfolding transitions and domain rear-rangements. These structural changes can be studied and enforced by single-molecule experiments. It is desirable to understand the underlying molecular interactions that drive experimentally observed conforma-tional transitions and determine measured forces. Molecular Dynamics simulations in combination with ad-vanced sampling approaches will be used to study unfolding/folding transitions in small model proteins under the influence of an external force and to investigate global domain motions in the Hsp90 chaperone protein. The simulations will allow us to characterize intermediates of protein folding/unfolding under force and to better understand the allosteric coupling of protein domain motion.
|
|
DNA's physical properties and packing in nucleosomes and higher order chromatin play critical roles in the storage, read out, and regulation of the genetic information. We propose to use massively parallel magnetic tweezers that enable to apply and directly measure physiologically relevant forces and torques at the single molecule level to dissect the mechanical properties and dynamics of nucleic acids and chromatin components. Building on this knowledge, we will determine how chromatin readers and remodelers alter the conformational landscape of nucleosomes and quantitatively test chromatin-remodeling mechanisms.
|
|
How the cellular DNA replication machinery overcomes obstacles encountered on chromosomes while avoiding damage and genome instability remains poorly understood. Here, we will study the dynamics of these events using single-molecule imaging approaches to directly observe the kinetics of replication during encounters with topological barriers. The results will clarify the influence of topological strain on replisome dynamics and the consequence of collisions between the DNA replication machinery and ring-shaped ATPases (SMCs) that play vital roles in chromosome organisation and segregation.
|
|
ATP-binding cassette (ABC) transporters are essential membrane proteins found in both eukaryotic and bacterial cells that facilitate the uphill transport of chemically diverse compounds. The molecular details of mechanochemical coupling of ATP-hydrolysis to conformational changes in those proteins remain to be elucidated. We here propose to study two ABC model systems with a combination of single-molecule fluorescence and force spectroscopy to characterize how the ATP-driven motor domain generates and transmits forces. With this we will unravel how chemical energy is converted into conformational changes of the pump domain that drive active transport.
|
|
The multiple tasks of the cytoskeleton rely on the tight spatial and temporal control of its hierarchical structure. In the proposed project, we plan to investigate how the conflicting functions of nucleation factors and crosslinking proteins lead to defined structural elements in reconstituted systems with increasing complexity. The major goal is to understand the interaction of reconstituted actin networks within lipid vesicles with the higher purpose of identifying the synergistic effects of the complexity of actin networks and lipid membranes. To address the composite nature of the cytoskeleton we plan to investigate the interaction of composite actin/keratin networks with lipid membranes.
|
|
This theoretical project aims at an understanding of molecular self-organization principles in cytoskelatal systems. We will apply and further develop agent-based computer simulations as well as coarse-grained continuum approaches to study how the combined action of mechanical forces and chemical kinetics orchestrates the formation of tightly regulated cytoskeletal architectures of well-defined size and shape as well as highly dynamic spatio-temporal patterns.
|
|
Integrins are signaling and force-transducing cellular receptors. They coordinate fundamental events by transducing forces from the cytoskeleton to the extracellular space and by translating external forces into biochemical signals within the cell. This exquisite property is required to orchestrate development of multicellular organisms, physiology, and pathology. The different members of the integrin family cooperate in force transmission and signaling. We propose to analyze integrin cooperativity under force using a combination of mouse genetics, whole cell proteomics and live-cell AFM.
|
|
Integration of the nucleus in the cytoskeleton and maintenance of nuclear integrity are important cellular activities. We want to investigate the molecular basis of the interaction between the nuclear envelope and cytoskeletal structures in the cytosol, and the impact of nuclear protein rods on nuclear mechanics. The main molecules of interest are the envelope proteins Sun-1 and the connection to the cytoskeleton via the nesprin-homologue interaptin, as well as a filamin/actin-like protein ('filactin') as component of nuclear rods. The work programme combines biophysical, biochemical and cell biological approaches.
|
|
We have identified a new type of isotropic acto-myosin network on the dorsal surface of cultured epithelial cells. To quantitatively understand the physical and biochemical properties of this unique network we will use live cell imaging in combination with mechanical and molecular perturbations of cells. This approach will enable us to bridge the gap between detailed physical models obtained in vitro and the much less understood complex cytoskeletal assemblies in living cells - one of the big challenges in the field of cytoskeleton mechanics.
|
|
The aim of this project is to understand the mechanics of motor proteins at the single molecule level and their cooperative organisation for specific cellular motile functions. We will apply a combination of bulk and single molecule techniques to study the mechanical properties of lipid-binding myosin motors in different oligomeric states. We propose to apply optical tweezers and single molecule fluorescence to study the mechanics of myosins class I, VI and XXI. The aim is to provide high-resolution single molecule data on regulated motor complexes to develop theoretical frame works of motor function in the cellular environment.
|
|
The mechanical properties of cytoskeletal filaments and networks are key for many vital processes in the cell. In this project, we plan to investigate cytoskeletal filament mechanics at the level of single filaments and filament-filament crosslinks. In the first part of this project using AFM force spectroscopy, we plan to characterize intermediate filaments and their building blocks from the level of coiled coil mechanics up to full filaments, in particular vimentin and desmin. In the second part of this project, we intend to close the gap between single filament mechanics and mechanics of large cytoskeletal networks using optical trapping.
|
|
Molecular motors take the lion’s share in organizing the cellular space by co-directing what, when, and where to transport intracellular cargo in eukaryotes. We dissect the working mechanisms of kinesin and myosin transporters at multiple levels to provide a molecular understanding of the underlying concepts of motor-dependent transport. Our aims are to understand (a) how the myosin-V dependent transport is regulated, (b) the interplay of myosin V-and kinesin-2 motors may benefit the intracellular transport of cargo, (c) how the side-stepping of kinesin-2 on the microtubule lattice may help the motor to efficiently navigate through road-blocks during cargo transport.
|
|
The critical role of mechanical forces for cell division has been recognized for decades. However, it is still unclear how force transmission at the kinetochore, a macromolecular complex linking microtubules and DNA, is mediated on the molecular level. In the last funding period we have established microscopy techniques and generated FRET-based biosensors to measure mechanical tension across distinct kinetochore proteins. Here, we propose to use these techniques to measure intracellular force transduction in living mitotic cells and to investigate force-dependent aspects of cell cycle regulation.
|
|
We want to quantitatively understand what forces are required to bend, bud, and generally transform cellular membranes. We will first establish a methodological platform that allows us to directly perform mechanical measurements on free-standing membranes. Second, we will specifically design membrane deforming molecules on basis of DNA origami, in order to study the influence of eminent factors, such as intrinsic molecular curvature and the propensity to build scaffolds through oligomerization and self-assembly.
|
|
We will study the complex rheological properties and self-healing behavior found in biological networks such as biofilms and cytoskeletal networks. A top-down approach is used that chemically modifies bacterial biofilm networks form various bacterial sources. Next to that, a bottom-up strategy will develop bioinspired polymer networks which are crosslinked by unique chemistry to tune the cross-linker density and their lifetime. The two types of networks will be tested for their complex rheological behavior and self-healing abilities in order to identify minimal combinations of macromolecules and cross-links that give rise to both strain-hardening and self-healing in the very same network.
|
|
Nucleosome positions within nucleosome arrays are determined by an interplay of nucleosome-nucleosome interactions, DNA sequence-dependent nucleosome stability, and active chromatin remodeling. Here, we study how the observed nucleosome gas statistics can robustly emerge from the underlying mechanisms, and how these mechanisms permit the rapid re-establishment of nucleosome arrays after they have been disrupted, e.g. during DNA replication. We will employ combinations of methods from statistical and soft-matter physics.
|
| Z |
Central Tasks of the SFB |
Prof. Dr. Mattias Rief
Physikdepartment der TUM, Lehrstuhl für Biophysik E22, München
|
to the top
|
 Events
23.05.2022
SFB Final Workshop 23./24. May 2022
This is our final workshop of the SFB 863 where we will discuss our recent scientific results. We look forward to meeting you after all the Corona lockdowns. >> read more
|
Seminars