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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), smallangle 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. FRETbased 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.protein folding | unfolded protein ensemble | Förster resonance energy transfer | nuclear magnetic resonance | small-angle X-ray scattering P roteins exist as ensembles of interconverting conformations.Obviously, unfolded polypeptide chains, such as chemically or physically denatured proteins and intrinsically disordered proteins (IDPs), can access extremely large numbers of conformations (1). A comprehensive description of their structural and dynamical behavior is a prerequisite for understanding protein folding (2-4) and the function of IDPs in health and disease (5, 6). Due to the very large number of conformational degrees of freedom of the unfolded polypeptide ensemble, it is of utmost importance to obtain as many independent experimental parameters as possible for a quantitative description of its behavior. Three experimental methods have been particularly informative in this respect: NMR spectroscopy (2, 5, 7-9), small angle X-ray scattering (SAXS) (10, 11), and single-molecule Förster resonance energy transfer spectroscopy (single-molecule FRET) (12, 13).NMR in solution provides very rich local structural and dynamical information at virtually any atom site with the exception of oxygen. Distance and angular information can be obtained from nuclear Overhauser enhancements (14), three-bond scalar couplings (15), paramagnetic relaxation enhancements (PREs) (16), pseudo contact shifts (17), residual dipolar couplings (RDCs) (8, 18), chemical shifts (19), and hydrogen bond scalar ...

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It is commonly believed that there are no detectable intermediates in the kinetic unfolding reactions of small proteins. If such intermediates could be found, they would give important information about the nature of the transition state for unfolding, which is thought to occur close to the native state. We report here that one-dimensional proton magnetic resonance spectra recorded during the unfolding of ribonuclease A provide direct evidence for at least one unfolding intermediate in which side chains are free to rotate. This intermediate appears to be a 'dry molten globule' of the kind hypothesized by Shakhnovich and Finkelstein.

1H, 13C, and 15N NMR assignments of the protein backbone of human interferon-gamma, a homodimer of 31.4 kDa, have been made using the recently introduced three-dimensional (3D) triple-resonance NMR techniques. It is shown that, despite the approximately 40-50-Hz 13C alpha and 1H alpha line widths of this high molecular weight dimer and the extensive overlap in the 1H alpha and 13C alpha spectral regions, unique sequential assignments can be made on the basis of combined use of the 3D HNCO, HNCA, HN(CO)CA, and HCACO constant-time experiments, the 15N-separated 3D NOESY-HMQC, and the 3D HOHAHA-HMQC experiments. Analysis of the 15N-separated 3D NOESY-HMQC and 13C/15N-separated four-dimensional (4D) NOESY-HMQC spectra together with the secondary C alpha and C beta chemical shifts yielded extensive secondary structure information. The NMR-derived secondary structure essentially confirms results of a recently published low-resolution crystal structure [Ealick et al. (1991) Science 252, 698-702], i.e., six helices in the monomer which are mostly alpha-helical in nature, no beta-sheets, a long flexible loop between helices A and B, and a very hydrophobic helix C. The functionally important carboxy terminus, which was not observed in the X-ray study, does not adopt a rigid conformation in solution. A high degree of internal mobility, starting at Pro-123, gives rise to significantly narrower resonance line widths for these carboxy-terminal residues compared to the rest of the protein.