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 ...

Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host.

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Switzerland Innovation, the Swiss innovation park with its five branches, is facilitating collaborations for companies, startups, and universities to find solutions to some of the world's most pressing challenges in the fields of health and the life sciences, in particular in the areas of chemistry, biochemistry, biomedicine, biotech, medtech and digital health. Together with its numerous and diverse partners, Switzerland Innovation creates an ecosystem for universities and research-based companies, accelerating the transformation of research results into marketable products and services.

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