hahn lab

RapR | UniRapR



Schematic representation of the approach used to regulate the catalytic activity of kinases. A fragment of FKBP is inserted at a position in the catalytic domain where it allosterically inhibits catalytic activity. Binding to rapamycin and FRB restores activity.


UniRapR (blue, subdomain-A; green, subdomain-B) protein was built by fusing FKBP and FRB. Using uniRapR there is no need to express two constructs. The fused FRB and FKBP, when inserted in the target protein, respond to rapamycin and activate it. This approach has been extended to apply to molecules other than kinases. See the "loopology" approach page.


Description of the approach

Karginov, A.V., Ding, F., Kota, P., Dokholyan, N.V., and Hahn, K.M. Engineered allosteric activation of kinases in living cells. Nature Biotech, July; 28(7):743-7, 2010. PMC2902629. Online article | Free PMC article

Karginov, A.V., Zou, Y., Shirvanyants, D., Kota, P., Dokholyan, N.V., Young, D.D., *Hahn, K.M.  and *Deiters, A. Light regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP. J. Am. Chem. Soc., 133: 420-423, 2011. PMC3133816. Online article | Free PMC article

Dagliyan, O., Shirvanyants, D., Karginov, A.V., Ding, F., Fee, L., Chandrasekaran, S.N., Freisinger, C.M., Smolen, G.A., Huttenlocher, A., *Hahn, K.M., *Dokholyan, N.V. Rational design of a ligand-controlled protein conformational switch. Proc. Natl. Acad. Sci. U.S.A., 110(17):6800-4, 2013. PMC3637791. Online article | Free PMC article


Additional applications

Chu, P-H., Tsygankov, D., Berginski, M.E., Dagliyan, O., Gomez, S.M., Elston, T.C., Karginov, A.V. and Hahn, K.M. Engineered kinase activation reveals unique morphodynamic phenotypes and associated trafficking for Src family isoforms. Proc. Natl. Acad. Sci. U.S.A.,111(34): 12420–12425, 2014. PMC4151743. Online article | Free PMC article

Dagliyan, O., Karginov, A.V., Yagishita, S., Gale, M.E., Wang, H., DerMardirossian, C., Wells, C.M., Dokholyan, N.V., Kasai, H. and Hahn, K.M. Engineering Pak1 Allosteric Switches. ACS Synth. Biol., 6 (7): 1257–1262, 2017. PMC5562282. Online article | Free PMC article (available July 21, 2018)


Detailed Methods

Karginov, A.V. and Hahn, K.M. Allosteric activation of kinases: design and application of RapR kinases. Curr. Protoc. Cell Biol., 53: 14.13.1 – 14.13.16, 2011. PMC3269071. Online article | Free PMC article

Gentry, L.R., Karginov, A.V., Hahn, K.M. and Der, C.J. Characterization of an engineered Src kinase to study Src signaling and biology. Methods Mol. Biol., 1360: 157-167, 2016. PMC4621786. Online article | Free PMC article

 

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