Sousa Francisco J R, Lima Luis M T R, Pacheco Ana B F, Oliveira Cristiano L P, Torriani Iris, Almeida Darcy F, Foguel Debora, Silva Jerson L, Mohana-Borges Ronaldo
Laboratório de Genômica Estrutural, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janerio, RJ, Brazil.
J Mol Biol. 2006 Jun 16;359(4):1059-74. doi: 10.1016/j.jmb.2006.03.069. Epub 2006 Apr 25.
Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.
研究了酸性pH对LexA阻遏物结构变化的影响。在pH 4.0左右会发生强烈的蛋白质聚集，但在低于pH 3.5的值时未检测到。相对于pH 7.2，Trp的光谱质量中心在pH 2.5时增加了400 cm(-1)，这表明LexA经历了结构重组但未变性。LexA在pH 2.5时的Trp荧光偏振表明其流体动力学体积大于其在pH 7.2时的二聚体。4,4'-二苯胺基-1,1'-联萘-5,5'-二磺酸（双ANS）实验表明，在中性pH时LexA结构中已存在的疏水裂缝中的残基在pH 2.5时对其具有更高的亲和力。当LexA在此pH下进行孔径限制的天然聚丙烯酰胺凝胶电泳时，获得了一条对应于四聚体的100 kDa条带。通过pH 2.5下的小角X射线散射（SAXS）分析也证实了这种四聚体状态的存在。一维1H NMR实验表明它由折叠和未折叠区域的混合物组成。尽管其稳定性比二聚体LexA低14000倍，但从静水压力和尿素曲线来看，它在pH 2.5时显示出四聚体-单体解离。尽管在pH 2.5时获得的亲和力是pH 7.2时的一半（Kaff为170 nM），但四聚体LexA在pH 2.5时仍能够结合recA操纵序列。此外，与在中性pH时不与阴性对照聚GC结合不同，LexA在pH 2.5时以1415 nM的Kaff值结合该序列。在pH 7.2和pH 2.5时进行的结合化学计量实验表明，[单体LexA]/[recA操纵序列]的比例为2:1。结合大肠杆菌SOS调节子在响应导致细胞内酸性pH的环境条件时的激活，对这些结果进行了讨论。此外，提出LexA的寡聚化是该调节子可能的调节机制。
