人组织型纤溶酶原激活剂重组kringle 2结构域的1H NMR结构表征。
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边IJ，凯利RF，利纳斯M
人组织型纤溶酶原激活剂重组kringle 2结构域的1H NMR结构表征。
生物化学。1989 11月28日;28(24):9350-60。
- PubMed ID
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2558718 (PubMed视图］
- 摘要
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人组织型纤溶酶原激活物(t-PA)的kringle 2结构域已在300和620 MHz下通过1H NMR光谱进行了表征。实验是在大肠杆菌中表达t-PA的174-263部分获得的分离结构域上进行的[Cleary et al. (1989) Biochemistry 28,1884 -1891]。t-PA kringle 2的光谱具有球状结构的特征，与纤溶酶原(PGN) kringle 4的光谱总体相似。与人类和牛PGN kringle 4的光谱比较确定了来自Leu46的侧链共振，它提供了kringle折叠的指纹，以及来自大多数芳香环自旋系统。t-PA kringle 2特有的His13、His48a和His64侧链产生的信号的分配通过His64----Tyr突变体的可用性得到帮助。配体结合研究证实，t-PA kringle 2与l -赖氨酸的结合常数Ka约为11.9 mM-1。这些数据表明，与PGN kringles 1和4的赖氨酸结合位点相关的同源或保守残基参与了l -赖氨酸与t-PA kringle 2的结合。这些基因包括Tyr36，以及kringle内环内的Trp62、His64、Trp72和Tyr74。在l -赖氨酸存在的情况下，芳香单线态的酸/碱滴定对His13和His64的pKa*分别约为6.25和4.41，并表明His48a咪唑基团不会质子化至pH*约为4.3。因此，His48a和His64侧链处于溶剂屏蔽位置。 As observed for the PGN kringles, the Trp62 indole group titrates with pKa* approximately 4.60, which indicates proximity of the side chain to a titratable carboxyl group, most likely that of Asp57 at the binding site. Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2H2O for full deuteration in the presence of L-lysine at 37 degrees C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1H2O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His48a imidazole NH3 proton and the three Trp indole NH1 protons. A strong dipolar interaction was observed among the Trp25 indole NH1, the Tyr50 amide NH, and the His48a imidazole CH2 protons, which affords evidence for an aromatic cluster in t-PA kringle 2 similar to that found at the hydrophobic kernel of PGN kringles.(ABSTRACT TRUNCATED AT 400 WORDS)