Luck L A, Falke J J
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1991 Jul 2;30(26):6484-90. doi: 10.1021/bi00240a019.
The Escherichia coli D-galactose and D-glucose receptor is a two-domain structure with a sugar-binding site at the interface between domains. The structure of the closed cleft containing bound D-glucose has been determined crystallographically, but the open cleft remains to be characterized. The present study illustrates a generalizable approach that is used to detect and analyze both the open- and closed-cleft conformations in solution. A 19F nucleus located inside the cleft is monitored by 19F NMR. When the cleft is occupied by D-glucose, the 19F nucleus is found to be inaccessible to the aqueous paramagnetic probe Gd-EDTA, verifying that the occupied cleft is closed in solution and inaccessible to bulk solvent. When the cleft is empty, the 19F nucleus becomes accessible to the paramagnet such that the distance of closest approach is r less than or equal to 10 A, indicating that the empty cleft opens at least transiently by an angle theta greater than or equal to 18 +/- 3 degrees.
大肠杆菌D-半乳糖和D-葡萄糖受体是一种双结构域结构，在结构域之间的界面处有一个糖结合位点。已通过晶体学确定了包含结合的D-葡萄糖的封闭裂隙的结构，但开放裂隙仍有待表征。本研究阐述了一种可推广的方法，用于检测和分析溶液中开放和封闭裂隙的构象。通过19F核磁共振监测位于裂隙内的19F原子核。当裂隙被D-葡萄糖占据时，发现19F原子核无法与顺磁水相探针Gd-EDTA接触，这证明被占据的裂隙在溶液中是封闭的，且无法与大量溶剂接触。当裂隙为空时，顺磁体可接触到19F原子核，使得最接近距离r小于或等于10埃，这表明空裂隙至少会短暂地打开一个角度θ，该角度大于或等于18±3度。
