Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the @entity1 @entity1372 / @entity413 cotransporter1 ( @entity67657 ) in @entity10056 and ligand-induced conformational changes of @entity67657 in solution as studied by intrinsic @entity2340 fluorescence. @entity67657 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB- @entity67657 was introduced into @entity10101 by electroporation. Purification of recombinant @entity67657 by @entity3406 -affinity chromatography yields about 3 mg of purified recombinant @entity67657 per 1-liter of cultured Pichia cells. Purified @entity67657 migrates on @entity214 -PAGE with an apparent mass of 55 kDa. Kinetic analysis of @entity67657 in proteoliposomes revealed @entity1372 -dependent, secondary active, @entity21146 -sensitive, and stereospecific @entity9786 transport, demonstrating its full catalytic activity. The position of the maximum intrinsic @entity2340 fluorescence and titration with hydrophilic collisional quenchers KI, @entity3323 , and @entity30728 suggested that most of Trps in @entity67657 in solution are in a hydrophobic environment. In the presence of @entity1372 , @entity3363 that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. @entity9786 had the highest affinity (K(d) = 0.71 mM), followed by @entity413 , @entity3649 , @entity3913 , and @entity69479 which showed a much lower affinity. @entity413 was without effect. @entity413 also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary @entity21146 , the well-established inhibitor of @entity67657 , decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were @entity1372 -dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of @entity67657 was labeled with @entity38263 , a reporter molecule for the surface hydrophobicity. In the presence of @entity1372 , addition of @entity413 decreased ANS fluorescence whereas @entity21146 increased ANS fluorescence. Thus three conformational states of @entity67657 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the @entity413 loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with @entity21146 .

High-yield functional expression of @entity1 @entity1372 / @entity413 cotransporter1 in XXXX and characterization of ligand-induced conformational changes as studied by @entity2340 fluorescence.

@entity1 :: ('9606', 'Species') :: ['human'] @entity3406 :: ('MESH:D009532', 'Chemical') :: ['nickel'] @entity3363 :: ('MESH:D002241', 'Chemical') :: ['sugars'] @entity10101 :: ('644223', 'Species') :: ['P. pastoris strain GS115'] @entity10056 :: ('4922', 'Species') :: ['Pichia pastoris'] @entity1372 :: ('MESH:D012964', 'Chemical') :: ['sodium'] @entity30728 :: ('MESH:C005849', 'Chemical') :: ['trichloroethanol'] @entity67657 :: ('6523', 'Gene') :: ['SGLT1', 'hSGLT1'] @entity2340 :: ('CHEBI:27897', 'Chemical') :: ['tryptophan'] @entity3323 :: ('MESH:D020106', 'Chemical') :: ['acrylamide'] @entity3913 :: ('MESH:D008358', 'Chemical') :: ['d-mannose'] @entity3649 :: ('MESH:D005690', 'Chemical') :: ['d-galactose'] @entity69479 :: ('CHEBI:40822', 'Chemical') :: ['d-allose'] @entity38263 :: ('MESH:C027132', 'Chemical') :: ['1-anilinonaphthalene-8-sulfonic acid'] @entity9786 :: ('MESH:C027020', 'Chemical') :: ['alpha-Methyl-d-glucopyranoside', 'alpha-methyl-d-glucopyranoside'] @entity413 :: ('MESH:D005947', 'Chemical') :: ['d-Glucose', 'l-Glucose', 'd-glucose'] @entity214 :: ('CHEBI:8984', 'Chemical') :: ['SDS'] @entity21146 :: ('MESH:D010695', 'Chemical') :: ['phlorizin']
@entity10056 :: (4922,Species) :: ['Pichia pastoris']
