Dataset Information

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

ABSTRACT: Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photo-catalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

SUBMITTER: Denis M

PROVIDER: S-EPMC7384118 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Publications

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

Denis Maxime M Softley Charlotte C Giuntini Stefano S Gentili Matteo M Ravera Enrico E Parigi Giacomo G Fragai Marco M Popowicz Grzegorz G Sattler Michael M Luchinat Claudio C Cerofolini Linda L Nativi Cristina C

Chemphyschem : a European journal of chemical physics and physical chemistry 20200319 9

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry ...[more]

PMID: 32092218

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Project description:Biomolecule-functionalized hydrogels have emerged as valuable cell culture platforms to recapitulate the mechanical and biochemical properties of the extracellular niche. The typical strategy to functionalize hydrogels with biomolecules involves directly tethering them to the hydrogel backbone resulting in a static material. Thus, this approach fails to capture the dynamic changes in biomolecule composition that occur during biological processes or that may be required for regenerative medicine applications. Moreover, it also limits the scope of biomolecules to simple peptides, as signaling proteins generally have poor stability under cell culture conditions and lose their bioactivity over time. To that end, we sought to develop a bioconjugation reaction that would enable reversible and repeatable tethering of signaling proteins to hydrogels, so that spent protein could be released on-demand and replaced with fresh protein as needed. Specifically, we designed an allyl sulfide chain-transfer agent that enables a reversible, photomediated, thiol-ene bioconjugation of signaling proteins to hydrogels. Upon addition of a thiolated protein to the allyl sulfide moiety, the previously tethered protein is released, and the "ene" functionality is regenerated. Using this approach, we demonstrate that protein patterning can be achieved in hydrogels through a thiol-ene reaction, and the patterned protein can then be released through a subsequent thiol-ene reaction of a PEG thiol. Importantly, this process is repeatable through multiple iterations and proceeds at physiologically relevant signaling protein concentrations. Finally, we demonstrate that whole signaling proteins can be patterned and released in the presence of cells, and that cells respond to their presentation with spatial fidelity. Combined, these data represent the first example of a methodology that enables fully reversible and repeatable patterning and release of signaling proteins from hydrogels.

Dataset Information

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

Publications

The Photocatalyzed Thiol-ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins.

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