Throughout the project, BIFOLDOME combines and develop advanced structural biology approaches to tackle the complexity of protein aggregation and amyloid formation. A major focus has been the structural characterization of proteins involved in necroptosis and neurodegenerative diseases, such as RIPK1 and RIPK3 as paradigm of RHIM-harboring proteins, as well as TDP-43.
A significant milestone was the successful production and stabilization of monomeric forms of these proteins, overcoming their natural tendency to aggregate during purification. By developing new detergent-based protocols and optimized conditions and co-solvents, the project is enabling a detailed characterization of the soluble forms of RIPK3, M45 and RIPK1 through solution NMR, shedding light on how unexpected specific regions drive amyloid formation with distinct secondary structure propensities. This led to the identification of a critical segment, the pre-RHIM region, as essential for initiating self-assembly, with different secondary structure patterns depending for each protein despite of adopting a conserved backbone fold in their corresponding amyloid states.
In parallel, solid-state NMR (SSNMR) and cryo-electron microscopy (cryo-EM) are being employed to resolve the structures of amyloid fibrils formed by RIPK1, RIPK3, and TDP-43. The installation of a CPMAS cryoprobe—one of only two in academic settings globally—is pivotal for these achievements. This technology enhances sensitivity to an unprecedented degree, enabling high-resolution spectra to be acquired in days rather than weeks, even with isotopically diluted samples. The structural resolution of RIPK1 fibrils marked the first full amyloid structure solved by SSNMR in Spain, positioning the laboratory as a reference point for amyloid research at the national level, while the installation of the singular CPMAS cryoprobe firmly establishes the lab on the international SSNMR landscape for the first time. These techniques are affording detailed characterization of distinct homo- and heteromeric amyloids.
Another critical development is OptoNMR, an optogenetic system that uses light to trigger and monitor protein assembly in real time within the NMR spectrometer. This method will allow for the controlled induction of phase transitions and fibril formation, providing insights into how proteins like TDP-43 transition from biomolecular condensates to amyloid fibrils.
Computational advances also played a key role in the project. BIFOLDOME is developing a web-based platform designed to predict amyloid interactions and calculate the energetic preferences of proteins to form homomeric or heteromeric assemblies. This tool significantly reduces the computational cost of amyloid modeling, lowering the required calculations from nearly 100 million to just over 7,000, without sacrificing accuracy. The server will be made publicly available, extending the project’s impact beyond the laboratory by providing accessible resources to the broader scientific community.
Lastly, the project contributed to the structural characterization of Nsp8, a SARS-CoV-2 protein that interacts with double-stranded RNA (dsRNA). Recent studies have shown that SARS-CoV-2 proteins can interact with TDP-43, influencing its assembly processes. In this context, our in vitro work on Nsp8 suggests it may play a role in modulating TDP-43 aggregation. This line of research has expanded the scope of BIFOLDOME, providing a unique model to study interactions between disordered proteins and structured biomolecules.
