| Autor: |
van Montfort, Bart A., Schuurman-Wolters, Gea K., Wind, Joyce, Broos, Jaap, Robillard, George T., Poolman, Bert |
| Zdroj: |
Journal of Biological Chemistry; April 2002, Vol. 277 Issue: 17 p14717-14723, 7p |
| Abstrakt: |
A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia colimannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)3-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning α-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys73). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys134), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys134becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for α-helix packing in the mannitol permease. |
| Databáze: |
Supplemental Index |
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