Coates, C; Kerruth, S; Helassa, N; Torok, K
(2019)
Kinetic mechanisms of fast glutamate sensing by fluorescent protein probes.
BIOPHYSICAL JOURNAL.
ISSN 0006-3495
https://doi.org/10.1016/j.bpj.2019.11.006
SGUL Authors: Torok, Katalin
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Abstract
We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGluh, iGlum and iGlul cover a broad range of Kd-s (5.8 μM, 2.1 mM and 50 mM, respectively) and a novel fluorescently labelled indicator, Fl-GluBP has a Kd of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerisation either of the apo or the ligand-bound binding protein. While the previously characterised ultrafast indicators iGluu and iGluf had monophasic fluorescence enhancement which occurred in the rate limiting isomerisation step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerisation steps. For iGlum and iGlul, the data indicate pre-binding conformational change followed by ligand binding. In contrast, for iGluh and Fl-GluBP glutamate binding is followed by isomerisation. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion limited rate constant to iGluh and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous AMPAR by glutamate binding.
Item Type: | Article | ||||||||||||
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Additional Information: | © 2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ | ||||||||||||
Keywords: | 02 Physical Sciences, 03 Chemical Sciences, 06 Biological Sciences, Biophysics | ||||||||||||
SGUL Research Institute / Research Centre: | Academic Structure > Molecular and Clinical Sciences Research Institute (MCS) | ||||||||||||
Journal or Publication Title: | BIOPHYSICAL JOURNAL | ||||||||||||
ISSN: | 0006-3495 | ||||||||||||
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Publisher License: | Creative Commons: Attribution-Noncommercial-No Derivative Works 4.0 | ||||||||||||
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URI: | http://sgultest.da.ulcc.ac.uk/id/eprint/111382 | ||||||||||||
Publisher's version: | https://doi.org/10.1016/j.bpj.2019.11.006 |
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