Assignment of 15N NMR signals in bovine pancreatic trypsin inhibitor
Assignment of 15N NMR signals in bovine pancreatic trypsin inhibitor
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牛胰腺胰蛋白酶抑制剂中 15N NMR 信号的归属
DOI：
10.1021/ja00259a045
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发表时间：
1987
影响因子：
15
通讯作者：
D. Cowburn
中科院分区：
文献类型：
--
作者：
J. Glushka;D. Cowburn
图1.绝对值·{15| BPTI在10%D2O/90%H2O，pH4.6，23mmol，68 ℃下的异频双量子光谱。所用的脉冲序列1是Rx（1H）-T-90 #o（15N）-r1 - 90，（15N）-Acquire #，-（），其中通常是Redfield 214选择脉冲25，r = 1/2J。质子载体频率为8.77 ppm（相对于内部三甲基丙酸参比）。数据矩阵为4096 X 128，t2和Zb的采集时间分别为680和16 ms。对于每个tx块，进行1200次累积，对于33 h的总累积时间，采集间延迟为100 ms。对t2应用高斯函数处理数据，并对t1中的512个点进行零填充。通过VNh偶联分裂的双重峰用线连接并用它们的分配标记。标记有星星的峰被认为属于缓慢交换的Asn 43和44羧酰胺。图中缺少来自D3、K15、G37、R39、K46和G57的信号。在其他温度下观察到D3、R39和K46; K15、G37和G57仍未指定。通过收集不同条件下的数据克服了重叠。在50 ° C和pH 3.5下，另外的酰胺和一些羧酰胺15N信号是可见的，并且可用的质子数据证实了先前的分配。在35 ℃，pH 4.6下，更多的信号是可见的，尽管缺乏详细的质子数据仅允许部分分配。在总共50的53个酰胺具有可观察到的质子被分配。17其中一个数据集的完整二维等高线图如图1所示。[18]根据对类似较小结构中13 C和15 N位移的广泛研究，可以合理地预期，距离最多4个键的原子会产生显著的取代基效应。19这些取代基效应可以被归为对氨基酰基残基的残基取代基效应和对前一个氨基酰基残基的最近邻效应。前者可以通过在DMSO中使用A-乙酰基氨基酸测量的值获得，这可能反映了侧链的主要作用。20 "22然后调整这些值以反映C14的相邻（17）位。由于质子重叠，E49和D3被认为是暂定的，并且在68处没有D3的信号。由于在68处的预期质子共振处不存在峰，因此未对K15和G57进行归属。在较低的温度下，K15峰仍然不明确，并且试验性的G57双峰不能与羧酰胺严格分离
Figure 1. Absolute value·{15| heteronuclear double quantum spectrum of BPTI in 10% DjO/90% H20, pH 4.6, 23 mmol, at 68 C. The pulse sequence1 used was Óx (1H)-T-90# o (15N)-r1-90,(15N)-Acquire#,-(), where was generally a Redfield 214 selective pulse25 and r= l/2J. The proton carrier frequency was at 8.77 ppm relative to internal trisylylpropionic acidreference. The data matrix was 4096 X 128 with acquisition times of 680 and 16 ms for t2 and Zb respectively. For each tx block, 1200 accumulations were taken, with an interacquisition delay of 100 ms for a total accumulation time of 33 h. The data were processed with a Gaussian function applied to t2 and zero filling to 512 points in t¡. Doublets split by VNh couplings are joined with a line and labeled with their assignment. Peaks labeled with a star are thought to belong to slowly exchanging Asn 43 and 44 carboxamides. Missingfrom this plot are signals from D3, K15, G37, R39, K46, and G57. D3, R39, and K46 were observed at other temperatures; K15, G37, and G57 remain unassigned. overlap were overcome by collecting data under different con-ditions. At 50 C and pH 3.5, additional amides and some carboxamide 15N signals were visible, and the proton data available corroborated previous assignments. At 35 C, pH 4.6, more signals were visible, though the lack of detailed proton data allowed for only partial assignments. In total 50 of the 53 amides having observable protons were assigned. 17 A complete 2D contour plotof one of the data sets is shown in Figure l. 18 In accordance with the extensive studies of 13C and 15N shifts in similar smaller structures, it is reasonable to expect significant substituent effects for atoms up tofour bonds away. 19 These substituent effects can be lumped into a residue substituent effect for the amino acyl residue and a nearest neighbor effect for the previous amino acyl residue. The former can be obtained from values measured by using A-acetyl amino acids in DMSO, which presumably reflect primary effect of side chains. 20" 22 These values are then adjusted to reflect neighboring (17) Assignments for C14. E49 and D3 are considered tentative, due to proton overlap, and the absence of signals for D3 at 68. No assignments were given to K15 and G57 due to absence of peaks at the expected proton resonance at 68. At lower temperatures, K15 peaks remained ambiguous, and tentative G57 doublets could not be rigorously separated from carboxamide