Analysis of Large-Scale Structural Changes in Proteins with focus on the Recovery Stroke Mechanism of Myosin II

The mechanisms through which proteins achieve their functional three-dimensional structure starting from a string of amino acids, as well as the manner in which the interactions between different structural elements are orchestrated to mediate function are largely unknown, despite the large amount of data accumulating from theoretical and experimental studies. One clear view emerging from all these studies is that function is a result of the intrinsic protein dynamics and flexibility, namely the motions of its well-defined structural elements and their ability to change their position and shape in space to allow large conformational transitions necessary for the function. Simulation techniques have been increasingly used over the past years in the endeavour to solve the structure-function puzzle as they have proven to be powerful tools to investigate the dynamics of proteins. However, extracting useful dynamical information from trajectories thus generated in order to draw functionally relevant conclusions is not always straight forward, especially when the protein function involves concerted movements of entire protein domains. This is due to the high dimensionality of the energy surface the proteins can explore. Therefore, a decrease in complexity is to be desired and can be achieved in principle by reducing the number of dimensions to the ones capturing only the dominant motions of the protein. To this purpose, in this thesis two different dimensionality reducing techniques, namely Principal Component Analysis and Sammon Mapping are applied and compared on four proteins that undergo conformational changes with different amplitudes and mechanisms. In particular, the present thesis tackles the large conformational change occurring during the recovery stroke of myosin, using these methods and rigidity analysis algorithms in the attempt to elucidate in atomic detail the structural mechanism underlying the function of this protein that couples ATP hydrolysis to the mechanical force needed to achieve muscle contraction. The results presented in this thesis show the successful applicability of certain dimensionality reducing methods to large conformational changes and their suitability in analyzing and dissecting dynamical transitions in computationally generated trajectories. The findings regarding the recovery stroke step in the myosin cycle are consistent with experimental data coming from mutational studies and confirm the previously postulated communication mechanism between the active sites of the protein, thus representing a major contribution to the field of molecular motors and a strong evidence of the importance of theoretical studies in complementing the experimental investigations

Understanding Enzyme Catalysis Using Computer Simulation

Enzymes catalyze biochemical reactions with remarkable specificity and efficiency, usually under physiological conditions. Computer simulation is a powerful tool for understanding enzyme catalytic mechanisms, particularly in cases where standard experimental techniques may be of limited utility. Here, we present an overview of the application of computer simulation techniques to understanding enzyme catalytic mechanisms. Examples using quantum chemical methods, as well as combined quantum mechanical/classical mechanical approaches, are provided

Efficient Communication Interfaces for Distributed Energy Resources

The IEC 61850 standard originally was developed for the substation automation. During the past years it was adapted for the integration of distributed energy resources into communication networks, however, with specific requirements. Many small and midsize manufacturers are using, as controllers, a big variety of different microprocessors with limited performances. Such controllers need an interface for IEC 61850 communication networks with a basic functionality which can be implemented with limited costs. Based on their experiences during the realization of an IEC 61850 communication stack, the authors propose ways to support these requirements. In particular, communication interfaces for photovoltaics systems and wind power plants are considered.</jats:p

Efficient Communication Interfaces for Distributed Energy Resources

The IEC 61850 standard originally was developed for the substation automation. During the past years it was adapted for the integration of distributed energy resources into communication networks, however, with specific requirements. Many small and midsize manufacturers are using, as controllers, a big variety of different microprocessors with limited performances. Such controllers need an interface for IEC 61850 communication networks with a basic functionality which can be implemented with limited costs. Based on their experiences during the realization of an IEC 61850 communication stack, the authors propose ways to support these requirements. In particular, communication interfaces for photovoltaics systems and wind power plants are considered.</p

Atomically detailed simulation of the recovery stroke in myosin by Milestoning

Myosin II is a molecular motor that converts chemical to mechanical energy and enables muscle operations. After a power stroke, a recovery transition completes the cycle and returns the molecular motor to its prestroke state. Atomically detailed simulations in the framework of the Milestoning theory are used to calculate kinetics and mechanisms of the recovery stroke. Milestoning divides the process into transitions between hyper-surfaces (Milestones) along a reaction coordinate. Decorrelation of dynamics between sequential Milestones is assumed, which speeds up the atomically detailed simulations by a factor of millions. Two hundred trajectories of myosin with explicit water solvation are used to sample transitions between sequential pairs of Milestones. Collective motions of hundreds of atoms are described at atomic resolution and at the millisecond time scale. The experimentally measured transition time of about a millisecond is in good agreement with the computed time. The simulations support a sequential mechanism. In the first step the P-loop and switch 2 close on the ATP and in the second step the mechanical relaxation is induced via the relay and the SH1 helices. We propose that the entropy of switch 2 helps to drive the power stroke. Secondary structure elements are progressing through a small number of discrete states in a network of activated transitions and are assisted by side chain flips between rotameric states. The few-state sequential mechanism is likely to enhance the efficiency of the relaxation reducing the probability of off-pathway intermediates