STAPHYLOCOCCAL NUCLEASE - SEQUENTIAL ASSIGNMENTS AND SOLUTION STRUCTURE
STAPHYLOCOCCAL NUCLEASE - SEQUENTIAL ASSIGNMENTS AND SOLUTION STRUCTURE
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DOI：
10.1021/bi00439a028
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发表时间：
1989-06-27
期刊：
影响因子：
2.9
通讯作者：
BAX, A
中科院分区：
文献类型：
--
作者：
TORCHIA, DA;SPARKS, SW;BAX, A
本文报道了葡萄球菌核酸酶（Nase）与胸苷3 ''，5 ''-二磷酸和Ca ~（2+）复合物的几乎所有残基的骨架15 N和1H的序列归属。由于Nase三元复合物Mr 18 K的相对较大，我们分配策略的关键要素是使用同位素编辑的二维NMR光谱，特别是15 N编辑的核奥弗豪瑟增强光谱（NOESY），15 N编辑的J相关光谱（COSY）和1H/15 N或1H/13 C杂原子多重量子位移相关光谱（HMQC）。这些实验，再加上更传统的NOESY，COSY，和home-Hartmann-Hahn光谱的天然丰度或氘化样品，产生的127个氨基酸残基的骨架分配的结构部分的蛋白质。使用NOESY数据，我们鉴定了三个螺旋结构域和几个β-与晶体结构中鉴定的二级结构密切对应的片。此外，许多长程NOESY连接被确定为与来自晶体结构的距离一致。在残基50附近的序列区域在溶液中似乎比在晶体中更灵活和无序。非常缓慢交换的酰胺质子是发现在cruystal结构中氢键键合的那些;然而，即使氢键键合的酰胺位于类似类型的规则二级结构中，例如， α-的螺旋，交换率大不相同。
Sequential assignments are reported for backbone 15N and 1H of nearly all residues of staphylococcal nuclease (Nase) complexed with thymidine 3'',5''-diphosphate and Ca2+. Because of the relatively large size of the Nase ternary complex, Mr 18K, the crucial element of our assignment strategy was the use of isotope-edited two-dimensional NMR spectra, particularly 15N-edited nuclear Overhauser enhancement spectroscopy (NOESY), 15N-edited J-correlated spectroscopy (COSY), and 1H/15N or 1H/13C heteronuclear multiple quantum shift correlation spectroscopy (HMQC). These experiments, together with the more conventional NOESY, COSY, and homonuclear Hartmann-Hahn spectra of natural abundance or deuteriated samples, yielded backbone assignments of 127 of the 136 residues in the structured part of the protein. Using the NOESY data, we identified three helical domains and several .beta.-sheets which were in close correspondence with secondary structure identified in the crystal structure. Moreover, many long-range NOESY connectivities were identified that were in agreement with distances derived from the crystal structure. The region of the sequence in the neighborhood of residue 50 appears to be more flexible and disordered in solution than in the crystal. Very slowly exchanging amide protons are those found to be hydrogen bonded in the cruystal structure; however, even hydrogen-bonded amides located within similar types of regular secondary structures, e.g., .alpha.-helices, exchange with greatly different rates.