Solid-state nuclear magnetic resonance (SSNMR) spectroscopy is used for the first time to examine the relative substrate-binding affinities of mutant forms of the Escherichia coli sugar transporter GalP in membrane preparations. The SSNMR method of (13)C cross-polarization magic-angle spinning (CP-MAS) is applied to five site-specific mutants (W56F, W239F, R316W, T336Y and W434F), which have a range of different sugar-transport activities compared to the wild-type protein. It is shown that binding of the substrate D-glucose can be detected independently of sugar transport activity using SSNMR, and that the NMR peak intensities for uniformly (13)C-labelled glucose are consistent with wild-type GalP and the mutants having different affinities for the substrate. The W239F and W434F mutants showed binding affinities similar to that of the wild-type protein, whereas the affinity of glucose-binding to the W56F mutant was reduced. The R316W mutant showed no detectable binding; this position corresponds to the second basic residue in the highly conserved (R/K)XGR(R/K) motif in the major facilitator superfamily of transport proteins and to a mutation in human GLUT1 found in individuals with GLUT1-deficiency syndrome. The T336Y mutant also showed no detectable binding; this mutation is likely to have perturbed helix structure or packing to an extent that conformational changes in the protein are hindered. The effects of the mutations on substrate-binding are discussed with reference to the putative positions of the residues in a 3D homology model of GalP based on the X-ray crystal structure of the E. coli glycerol-3-phosphate transporter GlpT.

中文翻译：

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通过固态NMR测定的野生型和突变形式的大肠杆菌糖转运蛋白GalP的相对底物亲和力。

固态核磁共振（SSNMR）光谱首次用于检查膜制剂中大肠杆菌糖转运蛋白GalP突变形式的相对底物结合亲和力。（13）C交叉极化魔角旋转（CP-MAS）的SSNMR方法应用于五个位点特异性突变体（W56F，W239F，R316W，T336Y和W434F），它们具有一系列不同的糖转运活性与野生型蛋白质相比。结果表明，使用SSNMR可以独立于糖的转运活性检测底物D-葡萄糖的结合，并且均匀（13）C标记的葡萄糖的NMR峰强度与野生型GalP和具有不同亲和力的突变体一致用于基材。W239F和W434F突变体显示出与野生型蛋白相似的结合亲和力，而葡萄糖结合对W56F突变体的亲和力却降低了。R316W突变体显示无可检测的结合。该位置对应于转运蛋白主要易化子超家族中高度保守的（R / K）XGR（R / K）基序中的第二个基本残基，并对应于患有GLUT1缺乏症的人的人GLUT1中的突变。T336Y突变体也未显示可检测的结合。这种突变很可能已经干扰了螺旋结构或堆积，从而阻碍了蛋白质的构象变化。参考E的X射线晶体结构，结合GalP的3D同源性模型中残基的假定位置，讨论了突变对底物结合的影响。