Secondary Structure and Oligomerization of the E. coli Glycerol Facilitator
详细信息 查看全文
- 作者：Darren M. Manley ; Mark E. McComb ; Hé ; lè ; ne Perreault ; Lynda J. Donald ; Harry W. Duckworth ; and Joe D. O'Neil
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：October 10, 2000
- 年：2000
- 卷：39
- 期：40
- 页码：12303 - 12311
- 全文大小：174K
- 年卷期：v.39,no.40(October 10, 2000)
- ISSN：1520-4995
文摘
The Major Intrinsic Proteins are found throughout the bacterial, plant, and animal kingdomsand are responsible for the rapid transport of water and other small, polar solutes across membranes. Thesuperfamily includes the aquaporins, the aquaglyceroporins, and the glycerol facilitators. We haveoverexpressed and purified the Escherichia coli inner membrane glycerol facilitator. Approximately 7.5mg of 95% pure protein is obtained from 1 L of Escherichia coli cells using immobilized metal affinitychromatography. Well-resolved matrix-assisted laser desorption ionization mass spectra were obtainedby solubilization of the protein in octyl-
-D-glucopyranoside (Mr = 33 650.3; error ~0.4%). Therecombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibitedby HgCl2. Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomericwhen solubilized with dodecyl--D-maltoside, octyl--D-glucopyranoside, and sodium dodecyl sulfate,but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomericspecies are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circulardichroism analysis shows that the protein is predominantly 
-helical. Helix content is significantly higherin protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for thefacilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.
-D-glucopyranoside (Mr = 33 650.3; error ~0.4%). Therecombinant glycerol facilitator is inserted into the bacterial inner membrane, is functional, and is inhibitedby HgCl2. Polyacrylamide gel electrophoresis suggests that the facilitator is predominantly monomericwhen solubilized with dodecyl--D-maltoside, octyl--D-glucopyranoside, and sodium dodecyl sulfate,but that it self-associates, forming soluble oligomers when urea is used during extraction. Similar oligomericspecies are demonstrated to exist in the bacterial membrane by chemical cross-linking experiments. Circulardichroism analysis shows that the protein is predominantly -helical. Helix content is significantly higherin protein prepared in the absence of urea (42-55%) than in its presence (32%). A possible role for thefacilitator oligomers in interactions with, and regulation of, the glycerol kinase is discussed.