van Nuland N A, Kroon G J, Dijkstra K, Wolters G K, Scheek R M, Robillard G T
BIOSON Research Institute, University of Groningen, The Netherlands.
FEBS Lett. 1993 Jan 2;315(1):11-5. doi: 10.1016/0014-5793(93)81122-g.
The region of the surface of the histidine-containing protein (HPr) which interacts with the A domain of the mannitol-specific Enzyme II (II(Amt1)) has been mapped by titrating the A-domain into a solution of 15N-labeled HPr and monitoring the effects on the amide proton and nitrogen chemical shifts via heteronuclear single quantum correlation spectroscopy (HSQC). Fourteen of the eighty-five HPr amino acid residues show large changes in either the 15N or 1H chemical shifts or both as a result of the presence of II(Amt1) while a further seventeen residues experience lesser shifts. Most of the residues involved are surface residues accounting for approximately 25% of the surface of HPr. Phosphorylation of HPr with catalytic amounts of Enzyme I (EI), in the absence of II(Amt1) resulted in chemical shift changes in a sub-set of the above residues; these were located more in the vicinity of the active site phospho-histidine. Phosphorylation of the HPr/II(Amt1) complex resulted in a HSQC spectrum which was indistinguishable from the P-HPr spectrum in the absence of II(Amt1) indicating that, as expected, the complex P-HPr/P-II(Amt1) does not exist even at the high concentrations necessary for NMR.
通过将甘露醇特异性酶II（II(Amt1)）的A结构域滴定到15N标记的含组氨酸蛋白（HPr）溶液中，并通过异核单量子相关光谱法（HSQC）监测对酰胺质子和氮化学位移的影响，已绘制出HPr表面与II(Amt1)的A结构域相互作用的区域。由于II(Amt1)的存在，八十五个HPr氨基酸残基中的十四个在15N或1H化学位移或两者上都有很大变化，另外十七个残基的位移较小。涉及的大多数残基是表面残基，约占HPr表面的25%。在没有II(Amt1)的情况下，用催化量的酶I（EI）对HPr进行磷酸化导致上述部分残基的化学位移发生变化；这些残基更多地位于活性位点磷酸组氨酸附近。HPr/II(Amt1)复合物的磷酸化产生了一个HSQC光谱，该光谱与没有II(Amt1)时的P-HPr光谱无法区分，这表明，正如预期的那样，即使在NMR所需的高浓度下，复合物P-HPr/P-II(Amt1)也不存在。
