Krebs D, Dahmani B, el Antri S, Monnot M, Convert O, Mauffret O, Troalen F, Fermandjian S
Département de Biologie Structurale, URA 147 CNRS, Institut Gustave Roussy, Villejuif, France.
Eur J Biochem. 1995 Jul 15;231(2):370-80. doi: 10.1111/j.1432-1033.1995.tb20709.x.
The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were examined by joint means of 1H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252-281 in c-Jun) is responsible for sequence-specific recognition of DNA. A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% alpha helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP-responsive element)-containing hexadecanucleotide, the alpha helix was stabilized to an extent of 20% at 20 degrees C or 35% at 2 degrees C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the alpha-helix content so that about 60% of alpha helix was found at a ratio of 75% trifluoroethanol (20 degrees C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helix/unordered transition. NMR data, namely alpha H chemical shifts, NOE cross-peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c-Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the alpha-helix structure mainly at these four sites. The malleability of the basic subdomain of c-Jun was emphasized in relation to its ability to fit the DNA helix in adopting an alpha-helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.
通过1H-NMR、圆二色（CD）和傅里叶变换红外（FTIR）光谱等联合手段，对碱性拉链（bZIP）蛋白c-Jun的碱性亚结构域的结构特性进行了研究。碱性亚结构域（c-Jun中的252-281位氨基酸残基）负责对DNA进行序列特异性识别。通过固相合成制备了修饰的碱性亚结构域bSD（1-35位氨基酸残基）及其N端部分和C端部分片段（NP，1-19位氨基酸残基；CP，16-35位氨基酸残基），并通过高效液相色谱（HPLC）进行纯化。在水溶液中，在不存在DNA的情况下，bSD主要表现为无结构的肽，其α螺旋含量仅为5%。然而，当将bSD与特定的DNA片段（即含CRE（cAMP反应元件）的十六聚体核苷酸）混合时，在20℃时α螺旋稳定至20%，在2℃时稳定至35%。同时，在DNA光谱中未检测到明显变化。向bSD水溶液样品中添加三氟乙醇导致α螺旋含量增加，在三氟乙醇比例为75%（20℃）时，约60%为α螺旋。这些效应在CD和FTIR测量中均有体现。该过程中CD光谱显示的变化表明其机制主要由双态螺旋/无序转变主导。核磁共振（NMR）数据，即αH化学位移、核Overhauser效应（NOE）交叉峰和NH温度系数，为c-Jun bSD序列中分散的四个短片段内的延伸或新生螺旋结构提供了线索，这与在水溶液中报道的酵母通用控制蛋白4（Gcn4）bSD独特且连续的螺旋片段形成对比。三氟乙醇主要在这四个位点稳定α螺旋结构。c-Jun碱性亚结构域在通过采用α螺旋结构来适配DNA螺旋的能力方面体现出了可塑性。复合物的形成显然需要肽发生大量构象变化，而寡核苷酸的构象变化很小。
