ausblenden:
Schlagwörter:
-
Zusammenfassung:
The attachment of the small protein ubiquitin (Ub) is a posttranslational modification that plays a key role in a vast array of cellular processes in eukaryotes. For the modification of a target protein / substrate a threeenzyme cascade is required: The Ub-activating enzyme (E1) activates the C-terminal glycine residue (G76) of Ub in an ATPdependent reaction. Afterwards, Ub is passed to the catalytic Cys of the Ub-conjugating enzyme (E2) and in the case of HECT-type ligases to the E3 itself through two consecutive transesterification-reactions. The HECT-type E3s then transfer Ub to the target protein resulting in a isopeptide linkage between the Ub C-terminus and the e-amino group of the lysine from the target protein. Our aim is to investigate the strucure of the HECT-Ub thioester which is formed in the Ub-transfer reaction from the E2 to E3. Structural studies of HECT thioester intermediates are aggravated by the inherent instability of the thioester bond. To overcome this problem we mimic the natural thioester linkage through a disulfide bond. For this, we form a disulfide bond between a C-terminal mutant of Ub (G76C) and the catalytic active cysteine of the HECT-domain. This approach allows us to study the intermediates of the HECT-Ub in more atomic detail by solution-state NMR spectroscopy and by X-ray crystallography.
