Tsao D H, Gruschus J M, Wang L H, Nirenberg M, Ferretti J A
Laboratory of Biophysical Chemistry, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
J Mol Biol. 1995 Aug 11;251(2):297-307. doi: 10.1006/jmbi.1995.0435.
We describe the NMR determination of the three-dimensional structure of a 77 amino acid residue protein, which consists of the 60 residue NK-2 homeodomain from Drosophila melanogaster and adjacent amino acid residues. The NK-2 homeodomain protein is part of a 723 amino acid residue protein which is expressed early in embryonic development in part of the central nervous system. NK-2 was characterized using both a natural abundance and a uniformly 15N enriched sample by two-dimensional and three-dimensional NMR experiments. The average root-mean-square deviation for 30 structures for residues 8 to 53 is 0.40 A for the backbone heavy-atoms and 0.72 A for the backbone and side-chain heavy-atoms. These structures were obtained from 986 NOE-derived upper and lower bound restraints. The three-dimensional structure contains three helices which consist of homeodomain amino acid residues 10 to 22, 28 to 38 and 42 to 52, as well as a turn between helix II and III, characteristic of homeodomains. Residues 53 to 60 of the DNA recognition helix are not fully ordered in the absence of DNA. In the free state this segment adopts a flexible but helix-like structure between residues 53 and 56 and is disordered from residues 57 to 60 although, as shown previously, the helix elongates by eight residues upon binding to DNA. The role of variable residues 52, 54 and 56 in determining the structure and flexibility of the recognition helix, as well as the stability of the NK-2 homeodomain as manifested by its thermal denaturation, are discussed.
我们描述了一种由77个氨基酸残基组成的蛋白质三维结构的核磁共振测定，该蛋白质由果蝇的60个残基NK-2同源结构域及相邻氨基酸残基组成。NK-2同源结构域蛋白是一种723个氨基酸残基蛋白质的一部分，该蛋白在胚胎发育早期于中枢神经系统的一部分中表达。通过二维和三维核磁共振实验，使用天然丰度样品和均匀15N富集样品对NK-2进行了表征。对于残基8至53的30个结构，主链重原子的平均均方根偏差为0.40 Å，主链和侧链重原子的平均均方根偏差为0.72 Å。这些结构是从986个源自核Overhauser效应（NOE）的上下限约束条件中获得的。三维结构包含三个螺旋，由同源结构域氨基酸残基10至22、28至38和42至52组成，以及螺旋II和III之间的一个转角，这是同源结构域的特征。在没有DNA的情况下，DNA识别螺旋的残基53至60没有完全有序排列。在自由状态下，该片段在残基53和56之间采用灵活但类似螺旋的结构，从残基57至60则无序，不过如先前所示，在与DNA结合时该螺旋会延长8个残基。讨论了可变残基52、54和56在确定识别螺旋的结构和灵活性以及NK-2同源结构域热变性所表现出的稳定性方面的作用。
