title: Comparative stability of Major Facilitator Superfamily transport proteins.
abstract: Membrane transporters are a vital class of proteins for which there is little available structural and thermodynamic information. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates in eukaryotes and prokaryotes. We have used far-UV circular dichroism (CD) to assess whether transporters from this superfamily have the same chemical and thermal stability. We have compared the stability of five different MFS transporters; PepTSofrom Shewanella oneidensis and LacY, GalP, GlpT and XylE from Escherichia coli, as well as a known stable mutant of LacY, LacY-C154G. CD stability measurements revealed that these transporters fall into two broad categories. The 'urea-sensitive' category includes LacY-WT, GalP and GlpT, which each lose around a third of their secondary structure in 8 M urea and two-thirds in the harsher denaturant guanidine hydrochloride (GuHCl). The 'urea-resistant' category includes LacY-C154G, XylE and PepTSo. These resistant transporters lose very little secondary structure in 8 M urea, and LacY-C154G and PepTSoresist denaturation by GuHCl up to a concentration of 4 M. The stabilities of LacY, GlpT, XylE and PepTSocorrelated with their crystal structure conformations, implying that a similar conformation is adopted in vitro. The 'urea-sensitive' transporters LacY and GlpT were crystallised inward-open states, while XylE and PepTSowere crystallised in occluded states. This study highlights the importance of studying a wide range of similar proteins, as a similar secondary structure and overall function does not necessarily confer the same stability in vitro.
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK.
authors.forename: Nicola J
authors.initials: NJ
authors.lastname: Harris
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Findlay
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sanders
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK.
authors.forename: Mateusz
authors.initials: M
authors.lastname: Kedzierski
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK.
authors.forename: Ália
authors.initials: Á
authors.lastname: Dos Santos
authors.affiliation: Department of Chemistry, King's College London, Britannia House, 7 Trinity Street, London, UK. paula.booth@kcl.ac.uk.
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Booth
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D000072417:Protein Domains; D055550:Protein Stability / Q000187:drug effects; D017433:Protein Structure, Secondary; D058767:Protein Unfolding / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D008024:Ligands; D026901:Membrane Transport Proteins; D014508:Urea; D019791:Guanidine
keywords: Circular dichroism; Major Facilitator Superfamily transporters