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Abstract: Understanding the function of a protein requires not only knowledge of its tertiary structure but also an understanding of its conformational dynamics. Nuclear magnetic resonance (NMR) spectroscopy, polarization-resolved fluorescence spectroscopy and molecular dynamics (MD) simulations are powerful methods to provide detailed insight into protein dynamics on multiple time scales by monitoring global rotational diffusion and local flexibility (order parameters) that are sensitive to inter- and intramolecular interactions, respectively. We present an integrated approach where data from these techniques are analyzed and interpreted within a joint theoretical description of depolarization and diffusion, demonstrating their conceptual similarities. This integrated approach is then applied to the autophagy-related protein GABARAP in its cytosolic form, elucidating its dynamics on the pico- to nanosecond time scale and its rotational and translational diffusion for protein concentrations spanning 9 orders of magnitude. We compare the dynamics of GABARAP as monitored by 15N spin relaxation of the backbone amide groups, fluorescence anisotropy decays and fluorescence correlation spectroscopy of side chains labeled with BODIPY FL, and molecular movies of the protein from MD simulations. The recovered parameters agree very well between the distinct techniques if the different measurement conditions (probe localization, sample concentration) are taken into account. Moreover, we propose a method that compares the order parameters of the backbone and side chains to identify potential hinges for large-scale, functionally relevant intradomain motions, such as residues 27/28 at the interface between the two subdomains of GABARAP. In conclusion, the integrated concept of cross-fertilizing techniques presented here is fundamental to obtaining a comprehensive quantitative picture of multiscale protein dynamics and solvation. The possibility to employ these validated techniques under cellular conditions and combine them with fluorescence imaging opens up the perspective of studying the functional dynamics of GABARAP or other proteins in live cells.

Abstract: Atomic models of proteins built by homology modeling or from low-resolution experimental data may contain considerable local errors. The refinement success of molecular dynamics simulations is usually limited by both force field accuracy and by the substantial width of the conformational distribution at physiological temperatures. We propose a method to overcome both these problems by coupling homologous replicas during a molecular dynamics simulation, which narrows the conformational distribution, and smoothens and even improves the energy landscape by adding evolutionary information.

Abstract: A novel protein refinement protocol is presented which utilizes molecular dynamics (MD) simulations of an ensemble of adaptively restrained homologous replicas. This approach adds evolutionary information to the force field and reduces random conformational fluctuations by coupling of several replicas. It is shown that this protocol refines the majority of models from the CASP11 refinement category and that larger conformational changes of the starting structure are possible than with current state of the art methods. The performance of this protocol in the CASP11 experiment is discussed. We found that the quality of the refined model is correlated with the structural variance of the coupled replicas, which therefore provides a good estimator of model quality. Furthermore, some remarkable refinement results are discussed in detail.