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Thermostabilisation of the serotonin transporter in a cocaine-bound conformation.
Abdul-Hussein, Saba; Andréll, Juni; Tate, Christopher G.
Afiliação
  • Abdul-Hussein S; MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
J Mol Biol ; 425(12): 2198-207, 2013 Jun 26.
Article em En | MEDLINE | ID: mdl-23706649
ABSTRACT
Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A+G278A+A505L) and SAH7 (L405A+P499A+A505L) that were more stable than SERT by 18°C and 16°C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [(125)I]RTI55 (ß-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [(3)H]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent Ki of 155µM or 82µM, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Cocaína / Proteínas da Membrana Plasmática de Transporte de Serotonina / Estabilidade Proteica Limite: Animals / Humans Idioma: En Revista: J Mol Biol Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Reino Unido

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Cocaína / Proteínas da Membrana Plasmática de Transporte de Serotonina / Estabilidade Proteica Limite: Animals / Humans Idioma: En Revista: J Mol Biol Ano de publicação: 2013 Tipo de documento: Article País de afiliação: Reino Unido