Periodic Reporting for period 1 - InProSpecT (Innovative Protein labelling strategies for Spectroscopic high resolution Techniques)
Período documentado: 2025-03-01 hasta 2027-02-28
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.
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.
We successfully synthesized the first generation of ultra-bright fluorophores, offering ready-to-use alternatives to traditional dyes. Specifically, we designed and synthesized deuterated, asymmetric rhodamines, which exhibit enhanced brightness and large Stokes shifts. During their preparation, we isolated atropisomeric dyes (stereoisomers resulting from restricted rotation around a single bond). While atropisomers have gained increasing attention in fields such as medicinal chemistry, catalysis, and molecular nanoscience, the development of atropisomeric fluorophores remains largely unexplored. Currently, we are leveraging these tools for single-molecule FRET (smFRET) studies of proteins such as AP180 and EPS15, as well as in other single-molecule fluorescence microscopy applications.
Recently, we demonstrated how the structure of atropisopure rhodamines can significantly enhance key photophysical properties. By integrating computational calculations and X-ray diffraction (DRX) analysis, we showed that these structural features are transferable to other fluorophore classes.
Single-molecule investigations are revolutionizing structural biology, yet intrinsically disordered proteins (IDPs); often found in phase-separated or complex environments, remain challenging to study using commercially available dyes. The tools we developed may represent a critical breakthrough in this field.
We anticipate that these dyes will have a transformative impact across biomedical and structural biology research. Our approach is highly adaptable, making it applicable to virtually any protein, in particular IDPs, and to other biomolecules, i.e. sugars (by click chemistry), lipids (by membrane insertion and photocrosslinking) and nucleotides (by aptamer binding). The novel insights gained into poorly studied proteins will not only stimulate scientific interest but also highlight the pivotal role of IDPs in addressing long-standing research questions. Ultimately, this work has the potential to influence drug development and make a meaningful impact on translational medicine.
Single-molecule investigations are revolutionizing structural biology, yet intrinsically disordered proteins (IDPs); often found in phase-separated or complex environments, remain challenging to study using commercially available dyes. The tools we developed may represent a critical breakthrough in this field.
We anticipate that these dyes will have a transformative impact across biomedical and structural biology research. Our approach is highly adaptable, making it applicable to virtually any protein, in particular IDPs, and to other biomolecules, i.e. sugars (by click chemistry), lipids (by membrane insertion and photocrosslinking) and nucleotides (by aptamer binding). The novel insights gained into poorly studied proteins will not only stimulate scientific interest but also highlight the pivotal role of IDPs in addressing long-standing research questions. Ultimately, this work has the potential to influence drug development and make a meaningful impact on translational medicine.