Characterization of the DNA Binding Properties of the Insulin Enhancer Binding Protein (Isl-1) Does the Zn Binding Domain Contribute to Stability and Specificity in DNA Binding?

DNA结合的特异性和稳定性取决于每个蛋白质/DNA复合体独特的结构、热力学和动力学性质。同源蛋白是一类定义明确的DNA结合蛋白，适用于许多不同的技术，旨在揭示蛋白质/核酸相互作用的本质。这些蛋白质是极其重要的转录因子，调节真核生物发育的关键阶段。本项目旨在为广泛的结构、动力学和热力学研究天然和重组ISL-1的DNA结合稳定性和特异性提供必要的基础。ISL-1是一种含有金属同源结构域(HD)的蛋白质，与大鼠胰岛素-I基因增强子结合并调节。本项目的目标是：1)从大肠杆菌表达菌株中纯化可溶性ISL-1；2)鉴定ISL-1：DNA结合的条件；3)通过过渡金属裂解(TMC)方法推导出DNA结合的一般溶液结构描述。这项STARTER赠款支持的初步表征将为设计更广泛的ISL-1 DNA结合研究提供基础。这样的研究很重要，因为它们增加了我们对过程的分子细节的理解，这些过程决定了为什么特定基因在发育过程中的特定时间表达。

9815327Frazee Both DNA binding specificity and stability depend upon structural, thermodynamic, and kinetic properties unique to each protein/DNA complex. Homeoproteins are an important class of well-defined DNA binding proteins that have been amenable to many and varied techniques designed to reveal the nature of protein/nucleic acid interactions. These proteins are extremely important transcription factors that regulate crucial stages in the development of eukaryotic organisms. This project seeks to provide the necessary foundation for extensive structural, kinetic, and thermodynamic studies of the DNA binding stability and specificity of natural and recombinant Isl-1, a metal-containing homeodomain (HD) protein that binds to and regulates the rat insulin-I gene enhancer. The objectives of the current project are: 1) to purify soluble Isl-1 from an E. coli expression strain; 2) to characterize Isl-1:DNA binding conditions; and 3) to deduce a general solution structure description of DNA binding via transitional metal cleavage (TMC) methodologies. The initial characterization supported by this starter grant will provide the foundation upon which more extensive studies on the Isl-1 DNA binding even may be designed. Such studies are important because they increase our understanding of the molecular details of processes that determine why specific genes are expressed at specified times during development.