Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching, Germany.
J Am Chem Soc. 2010 Nov 10;132(44):15692-8. doi: 10.1021/ja1064608.
Ph1500 is a homohexameric, two-domain protein of unknown function from the hyperthermophilic archaeon Pyrococcus horikoshii. The C-terminal hexamerization domain (Ph1500C) is of particular interest, as it lacks sequence homology to proteins of known structure. However, it resisted crystallization for X-ray analysis, and proteins of this size (49 kDa) present a considerable challenge to NMR structure determination in solution. We solved the high-resolution structure of Ph1500C, exploiting the hyperthermophilic nature of the protein to minimize unfavorable relaxation properties by high-temperature measurement. Thus, the side chain assignment (97%) and structure determination became possible at full proton density. To our knowledge, Ph1500C is the largest protein for which this has been achieved. To minimize detrimental fast water exchange of amide protons at increased temperature, we employed a strategy where the temperature was optimized separately for backbone and side chain experiments.
Ph1500 是一种来自嗜热古菌 Pyrococcus horikoshii 的同六聚体、双结构域未知功能蛋白。C 末端六聚化结构域（Ph1500C）特别有趣，因为它与已知结构的蛋白没有序列同源性。然而，它的 X 射线晶体结构分析受阻，并且对于溶液中的 NMR 结构测定，这种大小（49 kDa）的蛋白构成了相当大的挑战。我们利用该蛋白的嗜热性，通过高温测量最大限度地减少不利的弛豫特性，解决了 Ph1500C 的高分辨率结构问题。因此，在全质子密度下完成了侧链分配（97%）和结构测定。据我们所知，Ph1500C 是迄今为止实现这一目标的最大蛋白。为了最小化高温下酰胺质子的快速水交换对其造成的损害，我们采用了一种策略，分别优化了主链和侧链实验的温度。
