Transfer of stabilising mutations between different secondary active transporter families

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.

The Mycobacterium lipid transporter MmpL3 is dimeric in detergent solution, SMALPs and reconstituted nanodiscs

“…The first indication that MmpL3 was forming oligomeric arrangements came from SDS-PAGE analysis of full-length M. smegmatis MmpL3 (Ms-MmpL3) protein, when isolated in DDM both with/without reconstitution in nanodiscs (see Expanded View tuberculosis protein. In our hands we have observed higher molecular weight bands on SDS-PAGE gels that correspond to oligomeric forms of membrane transport proteins [44][45][46][47] , as confirmed by structural analysis. However, given the addition of SDS, and the heating that occurs during the electrophoresis process, it was not possible to rule out that these higher molecular weight bands are artefacts of the gel running process.…”

Structural and functional insights into the mechanism of action of plant borate transporters

“…The exception was A500R where the amounts of protein extracted into detergent (29%, Supplementary Table S2 ) were too low to be detectable following separation on the SEC column. In order to further characterise the individual proteins we also submitted them to hFSEC, heating the solubilised proteins to 46 °C (higher than the apparent T m for WT AtBOR1 fused with GFP 37 ) for 10 min prior to separation on the SEC column. Following heating the WT monodispersed protein peak height is substantially reduced with a concomitant increase in the size of the aggregation peak.…”