title: Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS.
abstract: The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland;
authors: Mikayel
authors: M
authors: Aznauryan
authors: Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors: Leonildo
authors: L
authors: Delgado
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland;
authors: Andrea
authors: A
authors: Soranno
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland;
authors: Daniel
authors: D
authors: Nettels
authors: Institute of Biochemistry and Molecular Biology, National Yang Ming University, Taipei City 112, Taiwan;
authors: Jie-Rong
authors: JR
authors: Huang
authors: Biozentrum, University of Basel, 4056 Basel, Switzerland;
authors: Alexander M
authors: AM
authors: Labhardt
authors: Biozentrum, University of Basel, 4056 Basel, Switzerland; stephan.grzesiek@unibas.ch schuler@bioc.uzh.ch.
authors: Stephan
authors: S
authors: Grzesiek
authors: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland; Department of Physics, University of Zurich, 8057 Zurich, Switzerland stephan.grzesiek@unibas.ch schuler@bioc.uzh.ch.
authors: Benjamin
authors: B
authors: Schuler
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry*; D053838:Scattering, Small Angle; D000072760:Single Molecule Imaging
chemical_list: D011506:Proteins
keywords: Förster resonance energy transfer; nuclear magnetic resonance; protein folding; small-angle X-ray scattering; unfolded protein ensemble