hahn lab

affinity reagent biosensors: sense endogenous proteins

(LEFT) This Cdc42 biosensor was constructed from a fragment of WASP that binds selectively to activated Cdc42 (due to the green CRIB domain). The WASP fragment was derivatized with an environment-sensing dye that changed fluorescence when the biosensor bound Cdc42. Dye at position 271 (red) was in a hydrophobic pocket formed by amino acids from both WASP (blue) and Cdc42 (orange). (RIGHT) A library of FN3 monobodies is screened to find a library member with the appropriate binding selectivity and affinity for the targeted protein state. The library is based on a uniform scaffold stable in living cells and suitable for conversion to biosensors. The appropriate library member is derivatized with an environmentally sensitive dye to report target binding. Here, binding to active Src family kinases leads to increased fluorescence from a merocyanine dye.

Description of the approach

Nalbant, P., L. Hodgson, V. Kraynov, A. Toutchkine, K.M. Hahn. Activation of Endogenous Cdc42 Visualized in Living Cells. Science, 305:1615-1619, 2004. Online article | PDF

MacNevin, C.J., Toutchkine, A., Marston, D.J., Hsu, C.W., Tsygankov, D., Li, L., Liu, B., Qi, T., Nguyen, D.V. and Hahn, K.M. Ratiometric imaging using a single dye enables simultaneous visualization of Rac1 and Cdc42 activation. J. Am. Chem. Soc., 138(8): 2571-2575, 2016. PMC4825053. Online article | Free PMC article

Additional sensors via affinity reagent screening

Gulyani, A., Vitriol, E., Allen, R., Wu, J., Gremyachinskiy, D., Lewis, S., Dewar, B., Graves, L.M., Kay, B.K., Kuhlman, B., Elston T. and Hahn, K.M.  A biosensor generated via high-throughput screening quantifies cell edge Src dynamics. Nature Chem. Biol., 7: 437-444, 2011. PMC3135387. Online article | Free PMC article

Kummer, L., Hsu, C.W., Dagliyan, O., MacNevin, C., Kaufholz, M., Zimmermann, B., Dokholyan, N.V., Hahn, K.M., Plückthun, A.  Knowledge-Based Design of a Biosensor to Quantify Localized ERK Activation in Living Cells.  Chem. Biol. 20(6): 847-56, 2013. PMC415710. Online article | Free PMC article

Detailed Methods

Hodgson, L. Nalbant, P. Shen, F., and Hahn, K. Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol., 406:140-156, 2006. Online article | PDF

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