Dekker N, Peters A R, Slotboom A J, Boelens R, Kaptein R, de Haas G
Center for Biomembranes and Lipid Enzymology, State University of Utrecht, The Netherlands.
Biochemistry. 1991 Mar 26;30(12):3135-46. doi: 10.1021/bi00226a022.
The solution structure of porcine pancreatic phospholipase A2 (124 residues, 14 kDa) has been studied by two-dimensional homonuclear 1H and two- and three-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Backbone assignments were made for 117 of the 124 amino acids. Short-range nuclear Overhauser effect (NOE) data show three alpha-helices from residues 1-13, 40-58, and 90-109, an antiparallel beta-sheet for residues 74-85, and a small antiparallel beta-sheet between residues 25-26 and 115-116. A 15N-1H heteronuclear multiple-quantum correlation experiment was used to monitor amide proton exchange over a period of 22 h. In total, 61 amide protons showed slow or intermediate exchange, 46 of which are located in the three large helices. Helix 90-109 was found to be considerably more stable than the other helices. For the beta-sheets, four hydrogen bonds could be identified. The secondary structure of porcine PLA in solution, as deduced from NMR, is basically the same as the structure of porcine PLA in the crystalline state. Differences were found in the following regions, however. Residues 1-6 in the first alpha-helix are less structured in solution than in the crystal structure. Whereas in the crystal structure residues 24-29 are involved both in a beta-sheet with residues 115-117 and in a hairpin turn, the expected hydrogen bonds between residues 24-117 and 25-29 do not show slow exchange behavior. This and the absence of several expected NOEs imply that this region has a less well defined structure in solution. Finally, the hydrogen bond between residues 78-81, which is part of a beta-sheet, does not show slow exchange behavior.
利用二维同核1H以及二维和三维异核15N-1H核磁共振光谱对猪胰磷脂酶A2（124个残基，14 kDa）的溶液结构进行了研究。已对124个氨基酸中的117个进行了主链归属。短程核Overhauser效应（NOE）数据显示，残基1-13、40-58和90-109处有三个α-螺旋，残基74-85处有一个反平行β-折叠，残基25-26和115-116之间有一个小的反平行β-折叠。利用15N-1H异核多量子相关实验监测了22小时内酰胺质子的交换情况。总共有61个酰胺质子表现出缓慢或中等程度的交换，其中46个位于三个大螺旋中。发现螺旋90-109比其他螺旋稳定得多。对于β-折叠，可以确定四个氢键。从核磁共振推导得出的溶液中猪PLA的二级结构与晶体状态下猪PLA的结构基本相同。然而，在以下区域发现了差异。第一个α-螺旋中的残基1-6在溶液中的结构比在晶体结构中少。在晶体结构中，残基24-29既参与了与残基115-117形成的β-折叠，又参与了发夹结构，但残基24-117和25-29之间预期的氢键并未表现出缓慢的交换行为。这以及几个预期的NOE的缺失意味着该区域在溶液中的结构定义不太明确。最后，作为β-折叠一部分的残基78-81之间的氢键并未表现出缓慢的交换行为。
