Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 176, Issue 3, Pages 395-403Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2011.09.012
Keywords
Atomic force microscopy; Membrane protein; Projection structure; Scintillation proximity assay; Transmission electron microscopy; Two-dimensional crystal
Funding
- Swiss National Foundation Scientific Research [31003A_125150]
- European Science Foundation [09-EuroSYNBIO-FP-012 NANOCELL]
- Bern University Research Foundation
- Novartis Foundation
- National Centre of Competence in Research (NCCR) TransCure
- Swiss National Science Foundation (SNF) [31003A_125150] Funding Source: Swiss National Science Foundation (SNF)
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The glucose transporter IICB of the Escherichia coli phosphotransferase system (PTS) consists of a polytopic membrane domain (IIC) responsible for substrate transport and a hydrophilic C-terminal domain (IIB) responsible for substrate phosphorylation. We have overexpressed and purified a triple mutant of IIC (mut-IIC), which had recently been shown to be suitable for crystallization purposes. Mut-IIC was homodimeric as determined by blue native-PAGE and gel-filtration, and had an eyeglasses-like structure as shown by negative-stain transmission electron microscopy (TEM) and single particle analysis. Glucose binding and transport by mut-IIC, mut-IICB and wildtype-IICB were compared with scintillation proximity and in vivo transport assays. Binding was reduced and transport was impaired by the triple mutation. The scintillation proximity assay allowed determination of substrate binding, affinity and specificity of wildtype-IICB by a direct method. 2D crystallization of mut-IIC yielded highly-ordered tubular crystals and made possible the calculation of a projection structure at 12 A resolution by negative-stain TEM. Immunogold labeling TEM revealed the sidedness of the tubular crystals, and high-resolution atomic force microscopy the surface structure of mut-IIC. This work presents the structure of a glucose PTS transporter at the highest resolution achieved so far and sets the basis for future structural studies. (C) 2011 Elsevier Inc. All rights reserved.
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