Photoactivatable Probes for Protein Labeling
Over the last decade, several protein tags have been developed for the specific and covalent labeling of fusion proteins with small organic probes. Among them, SNAP-tag has been used in many applications such as live cell imaging, protein microarrays and the study of protein-protein interactions. The reaction of SNAP-tag with the O6-benzylguanine (BG) derivative of a synthetic probe leads to the covalent attachment of the synthetic probe to a reactive cysteine residue of SNAP-tag. To broaden the applications of SNAP-tag, several photoactivatable molecules were designed, synthesized and used for different applications. In the first part of this thesis, caged BG derivatives are presented which display no reactivity towards SNAP-tag fusion proteins but can be efficiently uncaged by UV light. The reactivity of BG towards SNAP-tag was blocked by introducing a photocleavable group, 1-(2-nitrophenyl) ethyl (NPE), on the N7 position of the guanine moiety. The applicability of these caged derivatives in protein microarrays and light-induced dimerization of SNAP-tag fusion proteins was demonstrated. In the second part, we introduced a general strategy for the preparation of photoactivatable and photoconvertible fluorescent probes for labeling of SNAP-tag fusion proteins. The function of the probes is based on intramolecular fluorescence resonance energy transfer (FRET). This strategy permits to synthesize a photoactivatable version of any desired fluorophore provided that an appropriate quencher is available. In this approach, the BG-fluorophore derivative was linked to a quencher via a photocleavable linker. These photoactivatable fluorophores are weakly fluorescent prior to photoactivation and become highly fluorescent after UV irradiation. In addition, using the same strategy, photoconvertible fluorescent probes can be synthesized by replacing the quencher with a second fluorophore. We successfully synthesized photoactivatable fluorescein and Cy3 derivatives, and a photoconvertible Cy5-Cy3 probe. These probes were used for the study of the lateral mobility of SNAP-tag fusion proteins. In the third part, a novel caged rhodamine derivative and two caged TokyoGreen derivatives are presented. The main advantage of these molecules is that only one caging group is required to keep the fluorophore in a non-fluorescent state. The caged rhodamine derivative displayed a large fluorescence increase ( 200-fold) upon photoactivation and was used for the labeling of cell surface proteins. In the last part, two reversibly photoswitchable fluorescent probes were prepared which are applicable for stochastic optical reconstruction microscopy (STORM).
Keywords: protein labeling ; SNAP-tag ; photoactivation ; caged molecules ; caged fluorophores ; photoswitchable fluorophores ; marquage de protéines ; SNAP-tag ; photo-activation ; caged molécules ; caged fluorophores ; photoswitchable fluorophoresThèse École polytechnique fédérale de Lausanne EPFL, n° 4660 (2010)
Programme doctoral Chimie et Génie chimique
Faculté des sciences de base
Institut des sciences et ingénierie chimiques
Laboratoire d'ingénierie des protéines
Record created on 2010-02-11, modified on 2016-08-08