TALEs from a spring -- superelasticity of Tal effector protein structures

A simple force-probe setup is employed to study the mechanical properties of transcription activator-like effector (TALE) proteins in computer experiments. It is shown that their spring-like arrangement benefits superelastic behaviour which is manifested by large-scale global conformational changes along the helical axis, thus linking structure and dynamics in TALE proteins. As evidenced from the measured force-extension curves the dHax3 and PthXo1 TALEs behave like linear springs, obeying Hooke's law, for moderate global structural changes. For larger deformations, however, the proteins exhibit nonlinearities and the structures become stiffer the more they are stretched. Flexibility is not homogeneously distributed over TALE structure, but instead soft spots which correspond to the RVD loop residues and present key agents in the transmission of conformational motions are identified.Comment: 6 pages, 4 figure

Observing Dynamic Conformational Changes within the Coiled-Coil Domain of Different Laminin Isoforms Using High-Speed Atomic Force Microscopy

Laminins are trimeric glycoproteins with important roles in cell-matrix adhesion and tissue organization. The laminin alpha, ss, and gamma-chains have short N-terminal arms, while their C-termini are connected via a triple coiled-coil domain, giving the laminin molecule a well-characterized cross-shaped morphology as a result. The C-terminus of laminin alpha chains contains additional globular laminin G-like (LG) domains with important roles in mediating cell adhesion. Dynamic conformational changes of different laminin domains have been implicated in regulating laminin function, but so far have not been analyzed at the single-molecule level. High-speed atomic force microscopy (HS-AFM) is a unique tool for visualizing such dynamic conformational changes under physiological conditions at sub-second temporal resolution. After optimizing surface immobilization and imaging conditions, we characterized the ultrastructure of laminin-111 and laminin-332 using HS-AFM timelapse imaging. While laminin-111 features a stable S-shaped coiled-coil domain displaying little conformational rearrangement, laminin-332 coiled-coil domains undergo rapid switching between straight and bent conformations around a defined central molecular hinge. Complementing the experimental AFM data with AlphaFold-based coiled-coil structure prediction enabled us to pinpoint the position of the hinge region, as well as to identify potential molecular rearrangement processes permitting hinge flexibility. Coarse-grained molecular dynamics simulations provide further support for a spatially defined kinking mechanism in the laminin-332 coiled-coil domain. Finally, we observed the dynamic rearrangement of the C-terminal LG domains of laminin-111 and laminin-332, switching them between compact and open conformations. Thus, HS-AFM can directly visualize molecular rearrangement processes within different laminin isoforms and provide dynamic structural insight not available from other microscopy techniques

Structural heterogeneity of the ion and lipid channel TMEM16F

Transmembrane protein 16 F (TMEM16F) is a Ca2+-activated homodimer which functions as an ion channel and a phospholipid scramblase. Despite the availability of several TMEM16F cryogenic electron microscopy (cryo-EM) structures, the mechanism of activation and substrate translocation remains controversial, possibly due to restrictions in the accessible protein conformational space. In this study, we use atomic force microscopy under physiological conditions to reveal a range of structurally and mechanically diverse TMEM16F assemblies, characterized by variable inter-subunit dimerization interfaces and protomer orientations, which have escaped prior cryo-EM studies. Furthermore, we find that Ca2+-induced activation is associated to stepwise changes in the pore region that affect the mechanical properties of transmembrane helices TM3, TM4 and TM6. Our direct observation of membrane remodelling in response to Ca2+ binding along with additional electrophysiological analysis, relate this structural multiplicity of TMEM16F to lipid and ion permeation processes. These results thus demonstrate how conformational heterogeneity of TMEM16F directly contributes to its diverse physiological functions

Simulation atomic force microscopy to predict correlated conformational dynamics in proteins from topographic imaging

AbstractAtomic force microscopy (AFM) of proteins can detect only changes within the scanned molecular surface, missing all motions in other regions and thus information about functionally relevant conformational couplings. We show that simulation AFM can overcome this drawback by reconstruction of 3D molecular structures from topographic AFM images. A proof of principle demonstration is provided for an in-silico AFM experiment visualizing the conformational dynamics of a membrane transporter. The application shows that the alternating access mechanism underlying its operation can be retrieved from only AFM imaging of one membrane side. Simulation AFM is implemented in the freely available BioAFMviewer software platform, providing the convenient applicability to better understand experimental AFM observations.</jats:p

Strukturaufgelöste approximative Modellierung der Dynamik von Motorproteinen

Proteinmotoren sind komplexe Makromoleküle, die eine Vielzahl an dynamischen Prozessen in biologischen Zellen ausführen. Ihre Wirkungsweise beruht auf koordinierten zyklischen Änderungen ihrer Konformation die durch das Binden von ATP Molekülen und deren Hydrolyse hervorgerufen werden. Diese Operationszyklen können relativ langsam verlaufen und lassen sich deswegen im Allgemeinen nicht mit Hilfe detaillierter Methoden der Molekulardynamik untersuchen. In der vorliegenden Arbeit wird daher ein auf Approximationen beruhendes mechanisches Modell benutzt, welches Proteine als verformbare elastische Objekte begreift und deren Untersuchung in Modellrechnungen am Computer ermöglicht. Helikase-Motoren deren Funktion darin besteht sich entlang von Nukleinsäure-Strängen zu bewegen und deren Doppelhelix-Struktur aufzubrechen bilden den Schwerpunkt unserer Untersuchungen. Fuer eine bedeutende Helikase, die des Hepatitis C Virus, konnten wir durch unsere Simulationen die dabei angewandten Mechanismen erklären. Zum ersten Mal konnte somit die Aktivität eines wichtigen molekularen Motors strukturaufgelöst in dynamischen Simulationen nachvollzogen werden. Darüber hinaus konnten wir durch die Anwendung unseren Modells auf weitere Helikasen derselben Superfamilie 2 deren Konformationsdynamik untersuchen. Im letzten Teil der Arbeit haben wir Modellsysteme von synthetischen Motoren untersucht. Dazu haben wir einen Prototypen eines künstlichen Molekularmotors entworfen, der durch die Kopplung an ein Filament in der Lage war, seine internen Konformationsänderungen in Kräfte auf das Filament zu übersetzen um es zu transportieren. Hierbei wurden auch thermische Fluktuationen berücksichtigt. Die Funktionsweise dieses Modellmotors wurde an die des wichtigen Motorpoteins Myosin angelehnt, welches die Kontraktion in Muskeln kontrolliert.Motor proteins are complex macromolecules which have evolved through the biological evolution to carry out a variety of functions related to force generation and intracellular transport. Underlying their organized activity are ordered conformational motions induced by binding of ATP molecules and their hydrolysis. Since these cyclic conformational motions are slow, they cannot be reproduced in molecular-dynamics simulations with all-atom models. Therefore, coarse-grained descriptions of reduced complexity are needed. In this Thesis, a coarse-grained mechanical model, with a protein pictured as a deformable elastic network object, has been employed. The focus was on the investigations of helicase proteins which are molecular motors that translocate in a cell over nucleic acids and unwind their duplex structure. By using the coarse-grained dynamical description for the protein and DNA and including interactions with ATP molecules, we have successfully followed entire operation cycles of the hepatitis C virus (HCV) helicase, for which a large amount of experimental data is available. Thus, the operation of a real molecular motor could be reproduced - for the first time - in structurally resolved dynamical simulations. Additionally, conformational relaxation dynamics in three other helicases from the same superfamily 2 has been investigated through coarse-grained numerical simulations. In the last chapter of the Thesis, a different, but related problem is addressed. There, we construct and investigate an elastic-network model of a device that can be viewed as a prototype of artificial molecular motors. Similar to myosin motors responsible for force generation in the muscles, the designed machine is able to convert, through a ratchet mechanism, its active cyclic internal motions into a steady net force used to pull a filament. Thermal fluctuations are taken into account and artificial motor operation at different fluctuation levels is discussed

Nucleotide-Induced Conformational Dynamics in ABC Transporters from Structure-Based Coarse Grained Modeling

ATP-binding cassette (ABC) transporters are integral membrane proteins which mediate the exchange of diverse substrates across membranes powered by ATP molecules. Our understanding of their activity is still hampered since the conformational dynamics underlying the operation of such proteins cannot yet be resolved in detailed molecular dynamics studies. Here a coarse grained model which allows to mimic binding of nucleotides and follow subsequent conformational motions of full-length transporter structures in computer simulations is proposed and implemented. To justify its explanatory quality, the model is first applied to the maltose transporter system for which multiple conformations are known and we find that the model predictions agree remarkably well with the experimental data. For the MalK subunit the switching from open to the closed dimer configuration upon ATP binding is reproduced and, moreover, for the full-length maltose transporter, progression from inward-facing to the outward-facing state is correctly obtained. For the heme transporter HmuUV, for which only the free structure could yet be determined, the model was then applied to predict nucleotide-induced conformational motions. Upon binding of ATP-mimicking ligands the structure changed from aconformation in which the nucleotide-binding domains formed an open shape, to a conformation in which they were found in tight contact, while, at the same time, a pronounced rotation of the transmembrane domains was observed. This finding is supported by normal mode analysis, and, comparison with structural data of the homologous vitamin B12 transporter BtuCD suggests that the observed rotation mechanism may contribute a common functional aspect for this class of ABC transporters. Although in HmuuV noticeable rearrangement of essential transmembrane helices was detected, there are no indications from our simulations that ATP binding alone may facilitate propagation of substrate molecules in this transporter. Possible explanations are discussed in the light of currently debated transport scenarios of ABC transporters