Biophysical Society Thematic Meeting - June 28-July 1, 2015

New Biological Frontiers Illuminated by Molecular Sensors and Actuators

Poster Abstracts

16-POS Board 16 Development of New Fluorescent Proteins & Biosensors with Microfluidic Selection Systems Felix Vietmeyer 1 , Premashis Manna 1,2 , Kevin Dean 4 , Brett Fiedler 1,2 , Amy Palmer 2,3 , Ralph Jimenez 1,2 . 1 University of Colorado, Boulder, CO, USA, 2 University of Colorado, Boulder, CO, USA, 3 University of Colorado, Boulder, CO, USA, 4 University of Texas Southwest Medical Center, Dallas, TX, USA. Creation of new molecular sensors and actuators relies on methods for identifying complex photophysical phenotypes and subsequently performing separations on cell populations. We have developed microfluidic flow cytometry approaches tailored to interrogating the performance of genetically-encoded fluorophores and FRET sensors, and present results of studies employing these technologies. One of the systems rapidly screens cell-based FP libraries on the basis of multiple photophysical parameters relevant to imaging, including brightness, photostability, and excited-state lifetime (i.e. a proxy for fluorescence quantum yield). Molecular dynamics-guided design was used to create a library of mCherry mutants that was screened with this system, and resulted in identification of a variant with a higher stability β-barrel and improved photostability but reduced brightness due to reduction in the fluorescence quantum yield. To improve brightness, additional rounds of selection were performed by adding excited-state lifetime as a sorting criterion. The multiparameter sort identified multiple clones with improved photostability and up to double the excited-state lifetime of the parent mCherry FP. Some of the mutated positions were previously identified in molecular dynamics simulations as exhibiting large structural fluctuations. Subsequent rounds of screening were used to improve FP folding and maturation. A second instrument in our lab is tailored to the screening and development of FRET-sensors for quantifying cellular concentrations of specific analytes. It sorts cells at rates of dozens per second, based on the magnitude and kinetics of ligand-induced sensor response on timescales from milliseconds to 15 seconds. We used it to investigate several genetically-encoded FRET sensors, which show unexpected heterogeneity of response. We demonstrate identification and purification of responsive sub-populations of Zn 2+ sensors in HeLa cells.

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