Using the sugar transport protein, GalP, from Escherichia coli, which is a homologue of human GLUT transporters, we have overcome the challenges for achieving high-resolution [¹⁵N-¹H]- and [¹³C-¹H]-methyl-TROSY NMR spectra with a 52 kDa membrane protein that putatively has 12 transmembrane-spanning α-helices and used the spectra to detect inhibitor binding. The protein reconstituted in DDM detergent micelles retained structural and functional integrity for at least 48 h at a temperature of 25 °C as demonstrated by circular dichroism spectroscopy and fluorescence measurements of ligand binding, respectively. Selective labelling of tryptophan residues reproducibly gave 12 resolved signals for tryptophan ¹⁵N backbone positions and also resolved signals for ¹⁵N side-chain positions. For improved sensitivity isoleucine, leucine and valine (ILV) methyl-labelled protein was prepared, which produced unexpectedly well resolved [¹³C-¹H]-methyl-TROSY spectra showing clear signals for the majority of methyl groups. The GalP/GLUT inhibitor forskolin was added to the ILV-labelled sample inducing a pronounced chemical shift change in one Ile residue and more subtle changes in other methyl groups. This work demonstrates that high-resolution TROSY NMR spectra can be achieved with large complex α-helical membrane proteins without the use of elevated temperatures. This is a prerequisite to applying further labelling strategies and NMR experiments for measurement of dynamics, structure elucidation and use of the spectra to screen ligand binding.

使用来自大肠杆菌（人类GLUT转运蛋白的同源物）的糖转运蛋白GalP，我们克服了获得高分辨率[ ¹⁵ N- ¹ H]-和[ ¹³ C- ¹ H]-甲基- TROSY NMR光谱具有52 kDa的膜蛋白，该蛋白推测具有12个跨膜的α螺旋，并使用该光谱检测抑制剂的结合。DDM去污剂胶束中重构的蛋白质在25°C的温度下至少保持48 h的结构和功能完整性，分别通过圆二色光谱和配体结合的荧光测量证明。色氨酸残基的选择性标记可再现地提供色氨酸的12个已解析信号¹⁵ N个主链位置以及¹⁵ N个侧链位置的已解析信号。为了提高敏感性，制备了异亮氨酸，亮氨酸和缬氨酸（ILV）甲基标记的蛋白质，产生了出乎意料的良好分离[ ¹³ C- ¹H]-甲基-TROSY光谱显示大部分甲基的清晰信号。将GalP / GLUT抑制剂福司柯林添加到ILV标记的样品中，从而诱导一个Ile残基发生明显的化学位移变化，并导致其他甲基发生更细微的变化。这项工作表明，使用大型复杂的α-螺旋膜蛋白可以实现高分辨率的TROSY NMR光谱，而无需使用高温。这是应用进一步的标记策略和NMR实验测量动力学，结构阐明以及使用光谱筛选配体结合的前提。