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

54 / 88

50
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 Vietmeyer1, Premashis Manna1,2, Kevin Dean4, Brett Fiedler 1,2, Amy Palmer 2,3, Ralph
Jimenez1,2.
1University of Colorado, Boulder, CO, USA, 2University of Colorado, Boulder, CO, USA,
3University of Colorado, Boulder, CO, USA, 4University 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.