title: Stabilization of a prokaryotic LAT transporter by random mutagenesis.
abstract: 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.
authors.affiliation: Institute for Research in Biomedicine (IRB), Barcelona Institute of Science and Technology, 08028 Barcelona, Spain.
authors.forename: Arturo
authors.initials: A
authors.lastname: Rodríguez-Banqueri
authors.affiliation: Institute for Research in Biomedicine (IRB), Barcelona Institute of Science and Technology, 08028 Barcelona, Spain.
authors.forename: Ekaitz
authors.initials: E
authors.lastname: Errasti-Murugarren
authors.affiliation: Institute for Research in Biomedicine (IRB), Barcelona Institute of Science and Technology, 08028 Barcelona, Spain Spanish Biomedical Research Center in Rare Diseases (CIBERER), 08028 Barcelona, Spain.
authors.forename: Paola
authors.initials: P
authors.lastname: Bartoccioni
authors.affiliation: Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria 3052, Australia.
authors.forename: Lukasz
authors.initials: L
authors.lastname: Kowalczyk
authors.affiliation: Biophysics Unit, Department of Biochemistry and Molecular Biology, Autonomous University of Barcelona, 08193 Cerdanyola del Vallés, Spain.
authors.forename: Alex
authors.initials: A
authors.lastname: Perálvarez-Marín
authors.affiliation: Institute for Research in Biomedicine (IRB), Barcelona Institute of Science and Technology, 08028 Barcelona, Spain Spanish Biomedical Research Center in Rare Diseases (CIBERER), 08028 Barcelona, Spain Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain jl.vazquez.ibar@gmail.com manuel.palacin@irbbarcelona.org.
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
authors.affiliation: Institute for Integrative Biology of the Cell (I2BC), CEA, French National Centre for Scientific Research (CNRS) UMR 9198, University Paris-Sud, University Paris-Saclay, F-91198 Gif-sur-Yvette, France jl.vazquez.ibar@gmail.com manuel.palacin@irbbarcelona.org.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Vázquez-Ibar
mesh_terms: D019943:Amino Acid Substitution; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability
chemical_list: D026905:Amino Acid Transport Systems; D001426:Bacterial Proteins