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Pàgina inicial > Articles > Articles publicats > Stabilization of a prokaryotic LAT transporter by random mutagenesis |
Data: | 2016 |
Resum: | A fluorescence-based screen was used to analyze 70 LAT transporter mutants and identify variants with improved stability and monodispersity. 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 -serine/-threonine exchanger is the best-known prokaryotic paradigm of the mammalian -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. |
Ajuts: | Fundació la Marató de TV3 20132330 Ministerio de Ciencia e Innovación BFU2008-04637 Ministerio de Ciencia e Innovación SAF2015-64869-R Agència de Gestió d'Ajuts Universitaris i de Recerca 2014/SGR-298 |
Drets: | Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials i que es distribueixin sota la mateixa llicència que regula l'obra original. Cal que es reconegui l'autoria de l'obra original. |
Llengua: | Anglès |
Document: | Article ; recerca ; Versió publicada |
Publicat a: | The Journal of General Physiology, Vol. 147, Num. 4 (April 2016) , p. 353-368, ISSN 1540-7748 |
16 p, 1.9 MB |