Stabilization of a prokaryotic LAT transporter by random mutagenesis

Arturo Rodríguez-Banqueri, Ekaitz Errasti-Murugarren, Paola Bartoccioni, Lukasz Kowalczyk, Alex Perálvarez-Marín, Manuel Palacín, José Luis Vázquez-Ibar

Research output: Contribution to journalArticleResearchpeer-review

3 Citations (Scopus)

Abstract

© 2016 Rodríguez-Banqueri et al. The knowledge of three-dimensional structures at atomic resolution of membrane transport proteins has improved considerably our understanding of their physiological roles and pathological implications. However, most structural biology techniques require an optimal candidate within a protein family for structural determination with (a) reasonable production in heterologous hosts and (b) good stability in detergent micelles. SteT, the Bacillus subtilis l-serine/l-threonine exchanger is the best-known prokaryotic paradigm of the mammalian l-amino acid transporter (LAT) family. Unfortunately, SteT's lousy stability after extracting from the membrane prevents its structural characterization. Here, we have used an approach based on random mutagenesis to engineer stability in SteT. Using a split GFP complementation assay as reporter of protein expression and membrane insertion, we created a library of 70 SteT mutants each containing random replacements of one or two residues situated in the transmembrane domains. Analysis of expression and monodispersity in detergent of this library permitted the identification of evolved versions of SteT with a significant increase in both expression yield and stability in detergent with respect to wild type. In addition, these experiments revealed a correlation between the yield of expression and the stability in detergent micelles. Finally, and based on protein delipidation and relipidation assays together with transport experiments, possible mechanisms of SteT stabilization are discussed. Besides optimizing a member of the LAT family for structural determination, our work proposes a new approach that can be used to optimize any membrane protein of interest.
Original languageEnglish
Pages (from-to)353-368
JournalJournal of General Physiology
Volume147
Issue number4
DOIs
Publication statusPublished - 1 Jan 2016

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