Computational Modeling and Design of Specific Protein-DNA Interfaces

描述（由申请人提供）：蛋白质和DNA之间的序列特异性相互作用对于基因组信息的表达和维持至关重要。尽管全基因组测序项目产生了蛋白质-DNA相互作用的部分列表，直接以潜在结合位点的形式和间接以推断的蛋白质序列的形式，但这些数据并不直接说明这些部分之间的相互作用。我们的长期目标是开发一个定量模型，用于评估DNA结合蛋白的序列特异性结合偏好和亲和力。这项研究背后的具体假设是，蛋白质-DNA相互作用的准确建模和工程需要处理DNA和蛋白质的灵活性，以及物理上真实的评分功能。这是基于几项观察。首先，纯粹基于序列的模型在预测结合偏好方面仅部分成功。第二，结构数据表明，在蛋白质-DNA界面中的明确定义的位置处的单个氨基酸可以由于构象自由而参与上下文依赖的相互作用。因此，侧链的移动能力与基于序列的方法所做的共同假设相矛盾，即给定位置处的氨基酸具有单一作用模式。最后，同源蛋白质-DNA复合物的晶体结构的检查表明，蛋白质和DNA序列的变化伴随着适度但重要的结构重排。 该提案的具体目标是建立蛋白质-DNA相互作用的物理模型，并通过实验验证该模型的预测： (1)构建一个物理模型，描述蛋白质-DNA界面特异性的基础。 (2)为了生成一个模型，使用物理化学和统计力学的考虑，为双组分信号通路中发现的转录因子的翼螺旋家族的结合位点的预测。 (3)重新设计lAnil归巢核酸内切酶对冈比亚按蚊（疟疾的主要载体）中靶基因的DNA结合和切割偏好。

DESCRIPTION (provided by applicant): Sequence-specific interactions between proteins and DNA are critical for the expression and maintenance of genomic information. Although genome-wide sequencing projects yield a parts list for protein-DNA interactions, directly in the form of potential binding sites and indirectly in the form of inferred protein sequences, these data do not directly speak to the interactions between these parts. Our long-term goal is to develop a quantitative model for assessing the sequence-specific binding preferences and affinities of DNA-binding proteins. The specific hypothesis behind this research is that accurate modeling and engineering of protein-DNA interactions requires treatment of both DNA and protein flexibility, and physically realistic scoring functions. This is based on several observations. First, purely sequence-based models are only partially successful in predicting binding preferences. Second, structural data indicate that a single amino acid at a well-defined position in a protein-DNA interface can participate in context-dependent interactions due to conformational freedom. Thus, a side chain's ability to move contradicts the common assumption made by sequence-based methods that an amino acid at a given position has a single mode of action. Finally, examination of crystal structures of homologous protein-DNA complexes reveals that changes in protein and DNA sequences are accompanied by modest but significant structural rearrangements. The specific aims in this proposal are designed to generate a physical model for protein-DNA interactions and to test experimentally the predictions made by this model: (1) To construct a physical model for describing the basis for specificity in protein-DNA interfaces. (2) To generate a model, using physical chemical and statistical mechanical considerations, for the prediction of binding sites for the winged helix family of transcription factors found in two-component signaling pathways. (3) To redesign the DNA binding and cleavage preference of the lAnil homing endonuclease to target genes in Anopheles gambiae, the primary vector for malaria.