Project  2.3. Develop engineered protein domains and identify domain insertion sites to confer light-induced inhibition and rapamycin-induced activation of kinases and GEFs without sequestration. Our preliminary work has identified conserved sites in proteins where molecular movements are coupled to the movements of important parts of kinase and GEF active sites. We have engineered a domain whose molecular dynamics change upon rapamycin binding. Insertion of this domain (called the uniRapR domain) into the kinase site renders kinase activity rapamycin-sensitive. Related analyses are used to engineer protein inhibition by light, and to extend the technology to GEFs and ultimately other families of proteins.

http://www.hahnlab.com/tools/controlProt-SmMol.html

2-3-1-full

Figure 1 Design and generation of RapR-FAK. (a) Schematic representation of the approach used to regulate the catalytic activity of FAK. A fragment of FKBP is inserted at a position in the catalytic domain where it abrogates catalytic activity. Binding to rapamycin and FRB restores activity. (b) The truncated fragment of human FKBP12 (amino acids Thr22 through Glu108) inserted into the kinase domain. Blue and red, full-length FKBP12; red, proposed structure of the inserted fragment. The FKBP12 is shown in complex with rapamycin and FRB (cyan). (c) Immunoblot analysis of iFKBP interaction with rapamycin and FRB. Myc-tagged FKBP12 and iFKBP constructs were immunoprecipitated from cells treated for 1 h with either 200 nM rapamycin or ethanol (solvent control). Co-immunoprecipitation of co-expressed GFP-FRB was detected using anti-GFP antibody. (d) Changes in the molecular dynamics of iFKBP upon binding to rapamycin and FRB. Warmer colors and thicker backbone indicate increasing root mean square fluctuation.

Nature Biotech., 28(7): 743-7, 2010.

Curr. Protoc. Cell Biol., Chapter 14:Unit 14.13, 2011.