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.
