Deep mutagenesis, whereby tens of thousands of mutational effects are determined by combining in vitro selections of sequence variants with Illumina sequencing, is an emerging technology for indirectly interrogating and observing protein conformations in living cells. Using deep mutagenesis and molecular dynamics simulations to inform each other, we plan to determine the mechanism of sugar import by SWEET family transporters at atomic resolution. Fluorescent substrates have enabled us to use fluorescence-based sorting of libraries of transporter mutants to find mutations along the entire permeation pathway that increase or decrease substrate transport. These comprehensive mutational landscapes will be used to interpret and support/reject hypotheses from simulations, including the role of protein conformational change in substrate transport process, proposed free energy barriers in the conformational-free energy landscape that limit import kinetics, and how sugar transport process could shed light on the mechanisms of substrate selectivity in membrane transporters.

High-throughput experiments and simulations for investigating sugar transport in plants

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ACS Spring 2023

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