STRUCTURE AND DYNAMICS OF A DENATURED 131-RESIDUE FRAGMENT OF STAPHYLOCOCCAL NUCLEASE - A HETERONUCLEAR NMR-STUDY
STRUCTURE AND DYNAMICS OF A DENATURED 131-RESIDUE FRAGMENT OF STAPHYLOCOCCAL NUCLEASE - A HETERONUCLEAR NMR-STUDY
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DOI：
10.1021/bi00171a004
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发表时间：
1994-02-08
期刊：
影响因子：
2.9
通讯作者：
SHORTLE, D
中科院分区：
文献类型：
--
作者：
ALEXANDRESCU, AT;ABEYGUNAWARDANA, C;SHORTLE, D
通过删除149个残基的野生型蛋白质的残基4-12和141-149，获得了葡萄球菌核酸酶的部分折叠形式。序列特异性NMR共振分配已获得106的131个残基在此蛋白质片段中，通过使用多维三重共振NMR的样品富集C-13和N-15。对应于天然状态的螺旋2（残基98-106）和螺旋1（残基54-68）的残基产生螺旋结构特征的化学位移和NOE效应。然而，这些相同的残基，给出耦合常数和NOE效应，指示螺旋和扩展构象之间的快速构象平均。因此，在核酸酶片段中观察到的残留螺旋结构的持久性明显低于天然状态下的相应结构。基于H α化学位移，我们估计螺旋构象的分数人口为30%的螺旋2和10%的螺旋1。两个片段，83-86和94-97，显示NOE效应，耦合常数，降低酰胺温度系数与天然的反向转弯结构一致。C-末端α-螺旋以及5-链β-桶的第四和第五链几乎没有显示出有序结构的证据。β折叠的前三条链、催化环的一部分和螺旋3的第一圈给出的NMR数据明显比蛋白质的其余部分差，可能是交换增宽的结果，并且不能详细表征。片段中最持久的结构元件是天然的，这表明核酸酶可能通过分层机制折叠。
A partially folded form of staphylococcal nuclease has been obtained by deleting residues 4-12 and 141-149 of the 149-residue wild-type protein. Sequence-specific NMR resonance assignments have been obtained for 106 of the 131 residues in this protein fragment by using multi-dimensional triple resonance NMR of samples enriched with C-13 and N-15. Residues corresponding to helix 2 (residues 98-106) and helix 1 (residues 54-68) of the native state give chemical shifts and NOE effects characteristic of helical structure. These same residues, however, give coupling constants and NOE effects indicative of fast conformational averaging between helical and extended conformations. The residual helix structure observed in the nuclease fragment is thus considerably less persistent than the corresponding structure in the native state. Based on H alpha chemical shifts, we estimate the fractional population of helical conformers to be 30% for helix 2 and 10% for helix 1. Two segments, 83-86 and 94-97, show NOE effects, coupling constants, and lowered amide temperature coefficients consistent with a native-like reverse-turn structure. The C-terminal alpha-helix as well as the fourth and fifth strands of the 5-strand beta-barrel show little evidence for ordered structure. The first three strands of the beta-sheet, part of the catalytic loop, and the first turn of helix 3 give significantly poorer NMR data than the rest of the protein, possibly as a result of exchange broadening, and could not be characterized in detail. That the most persistent elements of structure in the fragment are native-like suggests that nuclease may fold by a hierarchical mechanism.