Fuzzy complexes represent one part of the currently on-going paradigm shift in structural biology, and understanding their mechanism of formation could lead to a fundamentally altered view of biomolecular recognition and new therapeutic prospects. By PhD Pétur Orri Heiðarsson, University of Zurich Single-Molecule Spectroscopy to Study Fuzzy Protein Complexes and Their Role in Chromatin Remodelling Intrinsically disordered proteins (IDPs) are abundant in the human proteome and often play important roles in signalling and regulation. These molecules do not possess the ability to fold into a stable and specific three-dimensional structure but instead consist of an ensemble of different structural states. Two IDPs can interact and form a complex where both partners remain unstructured, termed a fuzzy complex, but direct structural and mechanical evidence for their formation have remained limited. In this project we intend to study one example of such a fuzzy complex, formed by molecules involved in the regulation of the 3D-architecture of our genomes. To study such exceedingly complex and dynamic processes we employ state-of-the-art single-molecule spectroscopy to interrogate individual molecules, both in vitro and in living cells. Using these methods we intend to decipher the molecular mechanisms behind fuzzy complex formation and simultaneously elucidate its role in DNA remodelling. Fuzzy complexes represent one part of the currently on-going paradigm shift in structural biology, and understanding their mechanism of formation could lead to a fundamentally altered view of biomolecular recognition and new therapeutic prospects. Intrinsically Disordered Proteins and Fuzzy Complexes A central paradigm in structural biology, that the structure of proteins defines their function, has been seriously challenged due to the realisation that a significant fraction of all genomes codes for intrinsically disordered proteins (IDPs). These proteins do not under native conditions form a stable, well-defined tertiary structure but consist of an ensemble of different fluctuating conformational states. Predictions have shown that intrinsic disorder is enriched in regulatory and signalling proteins, and IDPs often act as molecular hubs that interact with a multitude of partners. Recently, evidence has accumulated for interactions where one or both disordered partners remain unstructured in the complex. Intrinsically disordered proteins (IDPs) Approximately 40 percent of the human genome codes for proteins that do not occupy a stable structure but instead interconvert between different conformations. These proteins, called intrinsically disordered proteins, often play important roles in cell regulation and signalling. These complexes, termed ‘fuzzy complexes’ (1), have been suggested to be an extreme yet possibly abundant example of the functional repertoire of IDPs. Fuzzy interactions may confer many advantages to proteins and enable highly diverse yet specific interactions. Disordered regions in a complex are also more accessible than ordered regions for chemical modifications, and thus preserving conformational plasticity in the complex may be beneficial by adding another layer of regulation. Although indirect evidence from binding studies has suggested some protein interactions to have properties of fuzziness, direct structural and mechanistic evidence have remained elusive. A detailed characterisation of unstructured complexes may prove central to understanding the role of IDPs within the complex framework of signalling in the eukaryotic interactome. Fuzzy Complexes in Chromatin Remodelling Chromatin remodelling is a fundamental process that governs the regulation of our genes. It is a process in which the accessibility of certain genes is altered to control when and to what extent these genes are translated into their respective protein sequences. Dynamic disorder has been suggested to play an important role for linker histone H1 function during chromatin remodelling (2). Histone H1 binds to and dynamically cycles between nucleosomes, the fundamental repeating unit of chromosomes where DNA is wrapped around an octamer of core histone proteins. H1 has extensive disordered regions that interact with the nucleosomal DNA but also interact with a number of proteins that induces conformational changes, allowing a sophisticated type of gene regulation that is poorly understood on the molecular level. The interaction of histone H1 with a highly negatively charged and disordered nuclear protein has been suggested to lead to the decondensation of nucleosome suprastructure (3). The two disordered proteins form a complex where significant biophysical evidence suggest that both components remain fully unstructured yet remarkably tightly bound. This particular interaction represents an extreme case of fuzziness and is therefore an ideal system to study the phenomenon from a mechanistic viewpoint. The long-term goal is to understand the role of histone H1 disorder dynamics in chromatin remodelling and ultimately decipher its effects on gene expression. Chromatin remodelling Chromatin remodelling is the dynamic process by which the architecture of chromosomes is changed by chemical modifications or binding of effector proteins. Structural variations of chromosomes alter the accessibility of genes to transcriptional regulators and ultimately controls gene expression. Clearing up a Fuzzy View, One Molecule at a Time This project is intended to study a complex and little understood phenomenon occurring during an inherently dynamic process central to gene regulation. Studying this type of complex molecular behaviour thus demands the use of sophisticated methodologies. Single-molecule spectroscopy is a technique that allows one to study individual proteins or nucleic acids, one molecule at a time (4). The technique makes it possible to look at the properties of a single molecule, instead of being limited by the average properties of millions of molecules, a problem frequently encountered with more traditional biophysical methods. By fluorescently tagging the biomolecule of interest and using laser optics to excite the fluorophore, we can detect the resulting photons that are emitted during the relaxation process. These photons contain a wealth of information and can be used to extract parameters describing the structure and dynamics of the biomolecule. For example, by attaching two fluorophores and taking advantage of a phenomenon called Förster resonance energy transfer, one can measure the distribution of distances between the two fluorophores and get a measure on the structural states that are populated by the protein molecule. We intend to gain a clear view of fuzzy complex formation and dynamics by using a wide variety of single-molecule spectroscopy experiments, both in the test tube and within living cells (5). Significance Experimental evidence and predictions from protein sequences suggest that many disordered complexes exist, which are amenable to detailed studies. From these studies, information can be extracted that may prove crucial, not only for understanding the individual proteins or underlying molecular mechanisms but for exploiting these and other systems as drug targets. In addition, mapping the structural determinants for fuzzy complex formation has large implications for the ability to predict and detect new disordered interactions. The information from single-molecule spectroscopy, possibly in combination with structural data from NMR spectroscopy, may be used as input for calculating structural ensembles with MD simulations, which can serve as platforms for structure-based design of molecules to target fuzzy complexes. Fuzzy interactions have also been suggested to be the driving force behind formation of hydrogel-like functional membrane-less organelles in the cell(6). Insights gained from study of fuzzy complexes may thus also be applicable to this recently recognised link between cellular function and structural organisation, and for the design of nano-materials with novel functions. Overall, global societal effects in terms of human health, treatments, and materials design can be reached from the unexplored realm of disordered protein complexes. About the Support from the Carlsberg Foundation The Carlsberg Foundation was pivotal in making this project a reality. The grant has allowed me to work at the forefront of biophysics, in the world-renowned single-molecule laboratory of Benjamin Schuler at the University of Zurich, and in the Structural Biology and NMR Laboratory at the University of Copenhagen. Initial results, obtained with the Carlsberg Foundation’s Internationalisation Fellowship, suggest that possibilities for taking the research to the next level are abundant. In general, the fellowship will allow me to gain significant expertise in single-molecule spectroscopy and subsequently establish my own research career within single molecule biophysics. References 1. Tompa P, Fuxreiter M (2008) Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions. Trends Biochem Sci 33(1):2–8. 2. Lu X, Hansen JC (2004) Identification of Specific Functional Subdomains within the Linker Histone H10 C-terminal Domain. J Biol Chem 279(10):8701–8707. 3. Gomez-Marquez J, Rodríguez P (1998) Prothymosin alpha is a chromatin-remodelling protein in mammalian cells. Biochem J 333 ( Pt 1:1–3. 4. Schuler B, Hofmann H (2013) Single-molecule spectroscopy of protein folding dynamics--expanding scope and timescales. Curr Opin Struct Biol 23(1):36–47. 5. König I, et al. (2015) Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells. Nat Methods (July). doi:10.1038/nmeth.3475. 6. Chen T, Song J, Chan HS (2015) Theoretical perspectives on nonnative interactions and intrinsic disorder in protein folding and binding. Curr Opin Struct Biol 30:32–42.