Investigation of molecular recognition properties of ubiquitin, polyubiquitin and a disease-associated mutant

The small protein ubiquitin acts as a versatile cellular signal that controls a wide range of biological processes. The specificity of ubiquitin signalling is achieved by alternative conjugation signals and interactions with ubiquitin-binding proteins. Despite tremendous advancements in our understanding of ubiquitin function, the molecular details of recognition are still not fully elucidated. In this regard, solution NMR spectroscopy studies show promise to shed light into transient molecular interactions and conformational dynamics governing ubiquitin-mediated signalling. The traditional methods of studying proteins are implemented within dilute solutions with less than 10 g/L of total protein concentration. This low concentration allows to obtain good signals but may not adequately represent a biological environment. One distinctive feature of cellular systems is that the cytoplasm is deeply crowded with macromolecules (50-400 g/L) which affect several protein attributes. Macromolecular crowding can result in non-specific interactions between the protein of interest and the target protein. The broad aim of our study is to understand the effects of macromolecular crowding on ubiquitin recognition. We focused on the ubiquitin-UBA interaction, investigating the perturbations induced by the presence of a synthetic crowding agent in comparison with dilute solution by NMR. We analysed differences in binding affinity, structure and dynamics of the complex dissolved in the different media. Protein-protein interactions are a prime target for drug development and chemical biology research. Mechanisms of protein recognition have been extensively studied for single-domain proteins, but are less well characterized for dynamic multidomain systems. PolyUb represent an important multidomain system that requires recognition by structurally diverse ubiquitin-interacting proteins. Thus, the development of chemical species able to selectively recognize polyUb has become a subject of strong interest. Clearly, nanoparticles (NPs) present several advantages for protein recognition, including a large surface available for interaction. In our project, we aimed to explore NP systems for the development of polyUb-specific receptors. We investigated the binding specificity of chemically diverse NPs towards structurally distinct polyUb. Solution NMR spectroscopy was chosen as the central experimental technique due to its ability to provide site-resolved information on reversibly binding protein-NP pairs. Our results constitute the basis for an improved understanding of polyUb recognition by artificial receptors and for the development of NP-based therapeutic strategies. Given the central role of the Ub network in cellular physiology, misregulation is often associated with diseases, including cancer, immune disorders, and neurodegeneration. Due to a possible participation of the frameshift Ub mutant Ubb+1 in the molecular events leading to neurotoxicity and neurodegeneration in Alzheimer’s disease, there is large interest in elucidating the structural details of this aberrant protein and the consequent functional differences with respect to the wild-type protein. In our work, we investigated structural and dynamic features of Ubb+1 using NMR methods that are particularly suited to explore protein molecules containing flexible domains such as the C-terminal extension of Ubb+1.