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?

9815327 Frazee DNA结合特异性和稳定性均取决于每种蛋白质/DNA复合物特有的结构、热力学和动力学性质。同源异型蛋白是一类重要的定义明确的DNA结合蛋白，其已经适用于许多不同的技术，旨在揭示蛋白质/核酸相互作用的性质。这些蛋白质是非常重要的转录因子，调节真核生物发育的关键阶段。该项目旨在为天然和重组Isl-1的DNA结合稳定性和特异性的广泛结构，动力学和热力学研究提供必要的基础，Isl-1是一种含金属的同源结构域（HD）蛋白，结合并调节大鼠胰岛素-I基因增强子。本课题的主要目的是：1）从大肠杆菌中分离纯化可溶性Isl-1; coli表达菌株; 2）表征Isl-1：DNA结合条件;和3）通过过渡金属裂解（TMC）方法推导DNA结合的一般溶液结构描述。由该启动补助金支持的初步表征将为设计更广泛的Isl-1 DNA结合研究奠定基础。此类研究很重要，因为它们增加了我们对过程分子细节的了解，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.