Integrated NMR, Fluorescence, and Molecular Dynamics
Benchmark Study of Protein Mechanics and Hydrodynamics
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Posted on 2019-01-24 - 00:47
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.
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Möckel, Christina; Kubiak, Jakub; Schillinger, Oliver; Kühnemuth, Ralf; Corte, Dennis Della; Schröder, Gunnar F.; et al. (2019). Integrated NMR, Fluorescence, and Molecular Dynamics
Benchmark Study of Protein Mechanics and Hydrodynamics. ACS Publications. Collection. https://doi.org/10.1021/acs.jpcb.8b08903