title: Binding of phlorizin to the C-terminal loop 13 of the Na(+)/glucose cotransporter does not depend on the [560-608] disulfide bond.
abstract: The disulfide bonds of the Na(+)/glucose cotransporter (SGLT1) are believed to participate in the binding of the transport inhibitor phlorizin. Here, we investigated the role of the [560-608] disulfide bond on the phlorizin-binding function of the C-terminal loop 13 of SGLT1 using 3-iodoacetamidophlorizin (3-IAP) as a probe. The reactivity of 3-IAP to the fully reduced loop 13 was competitively inhibited by phlorizin, as evident from the MALDI mass spectra. It indicates that the disulfide bond is not mandatory for phlorizin binding. CD and equilibrium unfolding studies showed that the secondary structure and conformation stability of loop 13 were not affected by removing the disulfide bond. Furthermore, we generated a series of loop 13 mutants to assess the contribution of the disulfide bond to phlorizin binding. A positive correlation between the stability and phlorizin affinity of the mutant proteins was observed, implying that the protein stability, rather than the disulfide bond, is relevant to the phlorizin-binding function of loop 13.
authors.affiliation: Beijing Institute of Infectious Diseases, Beijing 10039, PR China. xb_xia2001@yahoo.com
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Xia
authors.forename: Chiann-Tso
authors.initials: CT
authors.lastname: Lin
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.forename: Hongqing
authors.initials: H
authors.lastname: Fang
mesh_terms: D020106:Acrylamide / Q000378:metabolism; D003553:Cystine / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010695:Phlorhizin / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D051269:Sodium-Glucose Transporter 1; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea
chemical_list: D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D051269:Sodium-Glucose Transporter 1; D020106:Acrylamide; D003553:Cystine; D014508:Urea; D010695:Phlorhizin