Periodic Reporting for period 1 - BiFOLDOME (BiFoldome: Homo- and Hetero-typic Interactions in Assembled Foldomes)
Reporting period: 2022-11-01 to 2025-04-30
Amyloid fibrils are insoluble protein aggregates implicated in diseases such as Alzheimer’s and amyotrophic lateral sclerosis (ALS). However, they also play essential roles in normal physiological processes, including immune responses. Understanding how proteins transition from disordered, soluble states into structured, fibrillar assemblies is one of the major challenges in structural biology and biophysics.
The BIFOLDOME project aims to unravel the molecular mechanisms driving these transitions, focusing on proteins that are central to necroptosis – a form of programmed cell death – as well as proteins involved in ALS and other fatal diseases, where their ability to form biomolecular condensates and amyloids underlies both functional and pathological processes. These proteins assemble through regions that mediate self- and co-assembly, but the structural and mechanistic basis for this behavior remains poorly understood.
To address this, BIFOLDOME is developing integrative NMR-based techniques, supported by computational tools and complementary biophysical approaches, to investigate how intrinsically disordered regions (IDRs) drive the formation and evolution of amyloid fibrils and biomolecular condensates, and how these condensates transition into amyloids.
A key innovation of the project is OptoNMR – an optogenetic technique that uses light to trigger protein assembly in real time, allowing the study of amyloid formation and condensate maturation under physiological conditions. By mimicking cellular processes, this approach provides unprecedented insights into how proteins dynamically transition between disordered and fibrillar states.
In addition to advancing experimental methodologies, the project is creating open-access computational tools that predict and design amyloid binders, facilitating the exploration of amyloidogenic sequences. These tools will be valuable to the scientific community, with applications spanning biotechnology and biomedical research.
By bridging fundamental research with technological innovation, BIFOLDOME advances our understanding of amyloidogenesis, generating essential knowledge that could inform future biomarkers or therapeutic strategies for amyloid-driven diseases and inflammatory pathways. The project’s results are expected to provide a solid scientific foundation for developing novel treatments, while contributing to the broader exploration of protein aggregation processes.
The BIFOLDOME project aims to unravel the molecular mechanisms driving these transitions, focusing on proteins that are central to necroptosis – a form of programmed cell death – as well as proteins involved in ALS and other fatal diseases, where their ability to form biomolecular condensates and amyloids underlies both functional and pathological processes. These proteins assemble through regions that mediate self- and co-assembly, but the structural and mechanistic basis for this behavior remains poorly understood.
To address this, BIFOLDOME is developing integrative NMR-based techniques, supported by computational tools and complementary biophysical approaches, to investigate how intrinsically disordered regions (IDRs) drive the formation and evolution of amyloid fibrils and biomolecular condensates, and how these condensates transition into amyloids.
A key innovation of the project is OptoNMR – an optogenetic technique that uses light to trigger protein assembly in real time, allowing the study of amyloid formation and condensate maturation under physiological conditions. By mimicking cellular processes, this approach provides unprecedented insights into how proteins dynamically transition between disordered and fibrillar states.
In addition to advancing experimental methodologies, the project is creating open-access computational tools that predict and design amyloid binders, facilitating the exploration of amyloidogenic sequences. These tools will be valuable to the scientific community, with applications spanning biotechnology and biomedical research.
By bridging fundamental research with technological innovation, BIFOLDOME advances our understanding of amyloidogenesis, generating essential knowledge that could inform future biomarkers or therapeutic strategies for amyloid-driven diseases and inflammatory pathways. The project’s results are expected to provide a solid scientific foundation for developing novel treatments, while contributing to the broader exploration of protein aggregation processes.
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.
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.
The BIFOLDOME project is advancing our understanding of amyloid formation and biomolecular condensates. A pivotal achievement has been the establishment of solid-state NMR (SSNMR) with unprecedented sensitivity, made possible by the acquisition and implementation of a CPMAS cryoprobe—one of only two in academic laboratories worldwide. This technological milestone enabled the resolution of amyloid structures in record time, dramatically reducing data acquisition periods from weeks to just days. The ability to analyze heteromeric interfaces—typically diluted by nature—has set a new benchmark for SSNMR, positioning the project at the forefront of amyloid research.
In addition to experimental advancements, the development of computational tools designed to predict and rank the interaction energies of amyloidogenic sequences represents a significant leap forward. Traditional approaches would have required millions of calculations to evaluate amyloid propensities across different combinations of residues. The hybrid strategy employed by BIFOLDOME reduced this computational load by several orders of magnitude, providing a scalable and accessible platform for the design and analysis of amyloid interactions. These tools are expected to have broad applications in predicting assembling-prone sequences, aiding in the rational design of inhibitors or functional amyloids for biotechnology.
In addition to experimental advancements, the development of computational tools designed to predict and rank the interaction energies of amyloidogenic sequences represents a significant leap forward. Traditional approaches would have required millions of calculations to evaluate amyloid propensities across different combinations of residues. The hybrid strategy employed by BIFOLDOME reduced this computational load by several orders of magnitude, providing a scalable and accessible platform for the design and analysis of amyloid interactions. These tools are expected to have broad applications in predicting assembling-prone sequences, aiding in the rational design of inhibitors or functional amyloids for biotechnology.