InProSpecT · Innovative Protein labelling strategies for Spectroscopic high resolution Techniques
Horizon Europe — Marie Skłodowska-Curie Actions
- Duration
- 2025-03-01 → 2027-02-28
- EU contribution
- €173,847
- Participants
- 1
- Scheme
- HORIZON-TMA-MSCA-PF-EF
Lines connect the coordinator with its partners.
Results in brief
Innovative Protein labelling strategies for Spectroscopic high resolution Techniques
Determining the dynamics of proteins remains an unsolved challenge, hindering our ability to identify the specific functions and molecular mechanisms of protein systems. Despite groundbreaking advances in cryo-electron microscopy (cryo-EM) and modern structure prediction algorithms (e.g., AlphaFold), these methods struggle to resolve the structures of highly dynamic proteins, such as intrinsically disordered proteins (IDPs). Single-molecule Förster resonance energy transfer (smFRET) offers a promising solution. However, its success hinges on two critical factors: the choice of fluorophore and its covalent attachment to the protein. The ideal fluorophore should be bright, photostable, and compact, while the labeling strategy must ensure site-specific attachment without disrupting protein function. Cysteine labeling, though commonly used, poses challenges for proteins that rely on cysteines for disulfide bridges or metal coordination. Additionally, fluorescent background interference, particularly in cellular environments or complex systems like liquid-liquid phase-separated compartments (often formed by IDPs) demands the use of red-shifted fluorophores, where background noise is minimized. Unfortunately, current red fluorophores suffer from suboptimal brightness and biocompatibility. To address these limitations, this project called InProSpecT (Innovative Protein labelling strategies for Spectroscopic high-resolution Techniques) proposes a bottom-up approach: - Developing custom-tailored, precise, and user-friendly chemical biology probes, and - Pioneering innovative labeling strategies to elucidate IDP dynamics both in vitro and in cellulo. As a proof of concept, this strategy will be applied to proteins such as Eps15 and AP180, which are essential for clathrin-coated pit assembly during endocytosis. By resolving their molecular dynamics, this work will pave the way for understanding their fine-tuned interplay in health and disease.
Data: CORDIS, © European Union
Project objective
Protein structures, including their complexes, determine their specific function and the molecular working mechanisms. The astonishing developments in X-ray crystallography and cryo-EM allow insights into atomic levels of protein structures, however, commonly on their static, solid state conformations. Nuclear magnetic resonance (NMR) and single molecule fluorescence spectroscopy, on the other hand, allow observations of protein dynamics in solution, in real-time. In particular, specific intermediate and long-range interactions can be measured using paramagnetic resonance enhancements (PRE) or single molecule FörsterResonance Energy Transfer (smFRET), requiring site-specific labelling with paramagnetic or fluorescence probes, respectively.Naturally, the choice of the label and its covalent attachment to the protein are critical, however, traditional approaches rely on random cysteine labelling with only a handful of available probes, making measurements difficult to interpret and laborious. As such, there is an urgent demand to develop custom-tailored, selective, orthogonal, easy to use chemical biology probes with excellent spectroscopic properties (for smFRET and PRE) to drive the field of protein structure and dynamics. This proposal tackles current limitations in the peptide/protein labelling field by creating state-of-the-art fluorophores for red/far-red smFRET. Building on this, the best performing dyes will be unleashed to defined labelling of intrinsically disordered and native proteins, in vitro and in cellulo, respectively. Next, these concepts will be expanded to be merged with intramolecular PRE probes to combine to date mutually exclusive spectroscopic properties. InProSpecT (Innovative Protein labelling strategies for Spectroscopic high resolution Techniques) pushes a bottom-up approach, from fluorophore design and labelling strategies ultimately linking two worlds to allow unprecedented, simultaneous studies of protein distances and dynamics.
Original text from CORDIS.
Participants
- FORSCHUNGSVERBUND BERLIN EV · BerlinCoordinatorGermany
Links
- View on CORDIS
- DOI: 10.3030/101147198
- https://ec.europa.eu/research/participants/documents/downloadPublic?documentIds=080166e521670a80&appId=PPGMS
Data: CORDIS, © European Union