Identification of functionally important protein residues by means of entropy based methods, and experimental validation by mutational analysis

我们的第一代程序H2r通过基于熵的方法分析相关突变，以确定蛋白质中的关键残基位置。在目前的资助期内，H2r的预测已经通过对参与色氨酸生物合成的两种酶的突变分析进行了实验测试。事实证明，在确定的位置上的氨基酸交换改变了催化效率或蛋白质稳定性。在这些有希望的结果的基础上，我们希望进一步提高H2r的特异性和灵敏度。应通过用基于冯诺依曼熵的相关性度量取代香农熵来提高特异性。这使我们能够考虑残基对的物理和化学性质.相应的相似性矩阵在当前发现期间准备，并实施了第一个原型算法。如果H2r利用所有统计上显著的相关信号，则灵敏度将增加。一个特定于MSA的零模型和一个特定于残差对的方法来计算截止值即将完成，并将在未来几个月内集成。由此产生的算法将被用来确定一个更大的分数比迄今为止可能的个别蛋白质内的关键位置。除了预测单个蛋白质中的关键残基位置外，我们还计划将相关性分析扩展到蛋白质蛋白质复合物。这些位置将通过计算机模拟进行评估，并使用充分表征的酶和酶复合物通过突变实验验证预测。

Our first generation program H2r analyses correlated mutations by means of entropy-based methods in order to identify crucial residue positions in proteins. In the current funding period, predictions made by H2r have been experimentally tested by mutational analysis of two enzymes involved in the biosynthesis of tryptophan. It turned out that amino acid exchanges at the identified positions alter catalytic efficiency or protein stability. Building up on these promising results, we want to further increase specificity and sensitivity of H2r. Specificity shall be improved by replacing Shannon’s entropy with correlation measures based on the von Neumann entropy. This allows us to consider physical and chemical properties of residue- pairs. Corresponding similarity matrices were prepared during the current finding period and a first prototypic algorithm was implemented. Sensitivity will increase, if H2r exploits all statistically significant correlation signals. An MSA-specific null-model and a residue-pair-specific approach to compute a cut-off are nearly finished and will be integrated within the next months. The resulting algorithms will be utilized to identify a larger fraction of crucial positions within individual proteins than hitherto possible. In addition to predict crucial residue-positions in individual proteins, we plan to extend correlation analysis to proteinprotein complexes. These positions will be assessed in silico and predictions will be validated by mutational experiments using well characterized enzymes and enzyme complexes.