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Transfer of stabilising mutations between different secondary active transporter families
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Cecchetti, Cristina, Scull, Nicola J., Mohan, Thotegowdanapalya C., Alguel, Yilmaz, Jones, Alexandra M. C., Cameron, Alexander D. and Byrne, Bernadette (2021) Transfer of stabilising mutations between different secondary active transporter families. FEBS Open Bio, 11 (6). pp. 1685-1694. doi:10.1002/2211-5463.13168 ISSN 2211-5463.
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WRAP-transfer-stabilising-mutations-between-different-secondary-active-transporter-families-2021.pdf - Published Version - Requires a PDF viewer. Available under License Creative Commons Attribution 4.0. Download (2659Kb) | Preview |
Official URL: http://dx.doi.org/10.1002/2211-5463.13168
Abstract
Integral membrane transporters play essential roles in the movement of substrates across biological membranes. One approach to produce transporters suitable for structural studies is to introduce mutations that reduce conformational flexibility and increase stability. However, it can be difficult to predict which mutations will result in a more stable protein. Previously, we stabilised the uric acid‐xanthine transporter, UapA, a member of the SLC23 family, through introduction of a single‐point mutation, G411V, trapping the protein in the inward‐facing conformation. Here, we attempted to stabilise the structurally related BOR1 transporter from Arabidopsis thaliana, a member of the SLC4 family, by introducing the equivalent substitution. We identified possible residues, P362 and M363, in AtBOR1, likely to be equivalent to the G411 of UapA, and generated four mutants, P362V or L and M363F or Y. Stability analysis using heated Fluorescent Size Exclusion Chromatography indicated that the M363F/Y mutants were more stable than the WT AtBOR1 and P362V/L mutants. Furthermore, functional complementation analysis revealed that the M363F/Y mutants exhibited reduced transport activity compared to the P362V/L and WT proteins. Purification and crystallisation of the M363F/Y proteins yielded crystals that diffracted better than WT (5.5 vs 7 Å). We hypothesise that the increased bulk of the F and Y substitutions limits the ability of the protein to undergo the conformational rearrangements associated with transport. These proteins represent a basis for future studies on AtBOR1.
Item Type: | Journal Article | |||||||||||||||
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Subjects: | Q Science > QH Natural history Q Science > QP Physiology |
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Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) | |||||||||||||||
Library of Congress Subject Headings (LCSH): | Membrane proteins , Biological transport, Mutagenesis , Carrier proteins | |||||||||||||||
Journal or Publication Title: | FEBS Open Bio | |||||||||||||||
Publisher: | John Wiley & Sons Ltd. | |||||||||||||||
ISSN: | 2211-5463 | |||||||||||||||
Official Date: | June 2021 | |||||||||||||||
Dates: |
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Volume: | 11 | |||||||||||||||
Number: | 6 | |||||||||||||||
Page Range: | pp. 1685-1694 | |||||||||||||||
DOI: | 10.1002/2211-5463.13168 | |||||||||||||||
Status: | Peer Reviewed | |||||||||||||||
Publication Status: | Published | |||||||||||||||
Access rights to Published version: | Open Access (Creative Commons) | |||||||||||||||
Date of first compliant deposit: | 17 May 2021 | |||||||||||||||
Date of first compliant Open Access: | 18 May 2021 | |||||||||||||||
RIOXX Funder/Project Grant: |
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