Project description
Single-molecule fluorescence studies can provide valuable information about the structural dynamics, and therefore the function, of integral membrane transport proteins. This project aims to compare the functional dynamics of archaeal and human secondary active transporters using confocal and total internal fluorescence (TIRF) microscopy, to compare to optical tweezers data [1].
Objective: We seek to elucidate the timescales of the structural dynamics of essential secondary active transporters [2], [3] on the single-molecule level, and connect these to functional rates in bulk measurements under different conditions.
Significance: Transmembrane transport proteins are essential for regulating the uptake of vital molecules that cells cannot produce themselves. Understanding their functional dynamics in different environments can provide insights into cellular regulation and potential therapeutic targets.
Methodology: Using confocal fluorescence and TIRF microscopy, we will perform real-time measurements of the functional motions of secondary active transporters. We will then compare these rates to those measured in standard bulk assays under different conditions to obtain a clear picture of which variables determine their transport rates.
Expected Outcomes: We plan to gain an in-depth understanding of the differences in regulatory processes between archaeal and human transporters, shedding light on their evolutionary adaptations and functional mechanisms.
Bibliography:
[1] L. van der Sleen, J. A. Stevens, S. J. Marrink, B. Poolman, and K. Tych, “Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers,” Cell Rep, vol. 43, no. 4, p. 114110, Apr. 2024, doi: 10.1016/j.celrep.2024.114110.
[2] S. Jensen, A. Guskov, S. Rempel, I. Hänelt, and D. J. Slotboom, “Crystal structure of a substrate-free aspartate transporter,” Nat Struct Mol Biol, vol. 20, no. 10, pp. 1224–1226, Oct. 2013, doi: 10.1038/nsmb.2663.
[3] G. Trinco et al., “Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk,” Commun Biol, vol. 4, no. 1, pp. 1–11, Jun. 2021, doi: 10.1038/s42003-021-02267-y.
Additional specifications
We are seeking candidates with backgrounds in molecular biology, biochemistry or biophysics with experience in protein expression and purification protocols. Prior experience with biophysical methods, optical tweezers and/or with membrane proteins is not essential, but is highly relevant. Full training will be provided.
