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Cancer Research (Subject) / V.4 Targeted protein manipulation (Lesson)
- Explain the molecular mechanism of the LOV domain. How does UV light affect the conformational change? LOV domains contain a non-covalently bound flavin mononucleotide (FMN) chromophore in its dark-state form, and a Ser-Thr kinase. (LOV/Ja Helix closed) Upon blue-light absorption, FMN and the cystein residue form a covalent bond (reversible covalent cysteinyl addukt formation). Thereby a kinase is activated, leading to autophosphorylation and therefore a conformational change, exposing the effector binding domain (LOV/Ja Helix open).
- Explain the mechanism leading to the protein transport towards the plasmamembrane through FRB+FKBP chemical dimerization. The small molecule rapamycin binds FKBP (FK506 binding protein), which can bind another protein. Rapamycin binds the plasma localized protein FRB as well, which brings FKBP, FRB and the additional protein together at the plasma membrane.
- Which consequences can phosphorylation have on a protein? Phosphorylation can alter the... -activity -stability -localization -interactions ...of a protein.
- How can a molecule become caged? Explain also the process of uncaging. An amino acid becomes caged, when unnatural amino-acids are incorporated into the protein. Caged compounds are light-sensitive probes that functionally encapsulate biomolecules in an inactive form and is activated in the presence of a stimulating light source. To uncage molecules, a photolysis system is required to cleave the covalent bond. In most cases, photo-uncaging is the technique revealing the active region of a compound by the process of photolysis of the shielding molecule (‘cage’). Photostimulation methods fall into two general categories: 1) Using light to uncage a compound that then becomes biochemically active, binding to a downstream effector. 2) Using of light to activate a light-sensitive protein, which can then excite the cell expressing a desired molecule.
- On which 3 levels can we manipulate a proteins´function? 1) genomic level 2) mRNA level 3) protein level
- Name 2 general types of protein mutations that can affect a proteins activity 1) truncation 2) point mutation
- How would you find out if the kinase activity of a protein is important for a specific biological function? Performing a point mutation of the kinase to destroy its activity. Then investigating the biological function of interest whithout the kinase activity.
- What is the cellular consequence if a dominant active mutant version of the small GTPase Ras is overexpressed (such as in a cancer cell)? Enhanced proliferation or proliferation independent of signaling pathways.
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