Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation.
Variable contributions of tyrosine residues to the structural and spectroscopic properties of the factor for inversion stimulation.
复制标题
酪氨酸残基对反转刺激因子的结构和光谱特性的不同贡献。
DOI：
10.1021/bi035441k
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Colon,Wilfredo
中科院分区：
文献类型：
--
作者：
Boswell,Sarah;Mathew,John;Beach,Michael;Osuna,Robert;Colon,Wilfredo
酪氨酸残基在蛋白质中的不同作用可能归因于它们的双重疏水性和极性性质，这可以导致疏水和环堆积相互作用以及氢键。小的同源二聚体DNA结合蛋白，倒位刺激因子（FIS），含有四个酪氨酸残基位于位置38，51，69和95，每个参与特定的内或分子间的相互作用。为了研究它们对FIS的稳定性、灵活性和光谱性质的贡献，将每一个独立地突变为苯丙氨酸。平衡变性实验表明，Tyr 95和Tyr 51分别使FIS稳定约2和1 kcal/mol，这是它们参与氢键-盐桥网络的结果。相比之下，Tyr 38由于在疏水环境中放置羟基而使FIS不稳定约lkcal/mol。当暴露于溶剂的Tyr 69突变时，FIS的稳定性没有改变。胰蛋白酶和V8蛋白酶的有限蛋白水解分别用于监测C-末端（残基71 - 98）和二聚体核心（残基26 - 70）的柔性。Y 95 F和Y51 F FIS的结果显示，与C-末端相比，二聚体核心的蛋白水解敏感性不同，表明后者的柔性增加。各种FIS突变体的DNA结合亲和力仅受到适度影响，并且与胰蛋白酶蛋白水解探测的C-末端灵活性成反比。每个突变体的荧光贡献的反卷积显示，它在WT FIS中的每个酪氨酸的强度和方向不同，突出了特定的相互作用和局部环境在确定酪氨酸残基的荧光中的作用。在Y51 F和Y 95 F突变中观察到的稳定性、灵活性和信号的显著变化归因于它们在氢键-盐桥网络中的耦合参与。这些结果突出了酪氨酸氢键和包装相互作用的FIS的稳定性的重要性，并证明了不同的作用，酪氨酸残基可以发挥的结构和光谱特性，即使是小的蛋白质。
The diverse roles of tyrosine residues in proteins may be attributed to their dual hydrophobic and polar nature, which can result in hydrophobic and ring stacking interactions, as well as hydrogen bonding. The small homodimeric DNA binding protein, factor for inversion stimulation (FIS), contains four tyrosine residues located at positions 38, 51, 69, and 95, each involved in specific intra- or intermolecular interactions. To investigate their contributions to the stability, flexibility, and spectroscopic properties of FIS, each one was independently mutated to phenylalanine. Equilibrium denaturation experiments show that Tyr95 and Tyr51 stabilize FIS by about 2 and 1 kcal/mol, respectively, as a result of their involvement in a hydrogen bond−salt bridge network. In contrast, Tyr38 destabilizes FIS by about 1 kcal/mol due to the placement of a hydroxyl group in a hydrophobic environment. The stability of FIS was not altered when the solvent-exposed Tyr69 was mutated. Limited proteolysis with trypsin and V8 proteases was used to monitor the flexibility of the C-terminus (residues 71−98) and the dimer core (residues 26−70), respectively. The results for Y95F and Y51F FIS revealed a different proteolytic susceptibility of the dimer core compared to the C-terminus, suggesting an increased flexibility of the latter. DNA binding affinity of the various FIS mutants was only modestly affected and correlated inversely with the C-terminal flexibility probed by trypsin proteolysis. Deconvolution of the fluorescence contribution of each mutant revealed that it varies in intensity and direction for each tyrosine in WT FIS, highlighting the role of specific interactions and the local environment in determining the fluorescence of tyrosine residues. The significant changes in stability, flexibility, and signals observed for the Y51F and Y95F mutations are attributed to their coupled participation in the hydrogen bond−salt bridge network. These results highlight the importance of tyrosine hydrogen-bonding and packing interactions for the stability of FIS and demonstrate the varying roles that tyrosine residues can play on the structural and spectroscopic properties of even small proteins.