Jia X, Reisman J M, Hsu V L, Geiduschek E P, Parello J, Kearns D R
Department of Chemistry, University of California at San Diego, La Jolla 92093.
Biochemistry. 1994 Jul 26;33(29):8842-52. doi: 10.1021/bi00195a028.
Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.
已报道了转录因子1（TF1）的序列特异性1H和15N NMR1归属，TF1是一种22 kDa的II型DNA结合蛋白（DBPII），由两个99个残基的单体组成。采用了一种归属策略，该策略使用六种互补的选择性氘代TF1变体和一种均匀15N标记的TF1变体。对二维和三维同核和异核NMR相关谱进行了分析，得出了1H和15N共振的几乎完整的归属。还定义了离散的蛋白质二级结构域；在每个单体中，鉴定出三个α-螺旋、一个反平行β-折叠和一个反平行β-带。对由两种不同的选择性氘代单体形成的两个二聚体的分析，有助于确定许多质子间接触是单体间的还是单体内的。酰胺质子交换分析表明，每个单体中羧基末端的α-螺旋比其他两个α-螺旋稳定性差。基于高度同源的DBPII（嗜热脂肪芽孢杆菌编码的HU蛋白）的晶体结构，先前提出的TF1二聚体的工作结构模型[Geiduschek等人（1990年）《结构生物学杂志》104，84-90]，总体上得到了我们结果的支持。然而，注意到与该模型有一些偏差。最显著的是，TF1的羧基末端尾巴采用α-螺旋构象，在Lys93处主链发生扭曲。
