ALLOSTERIC DETERMINANTS IN THE LACI/GALR FAMILY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。蛋白质的序列和结构分析表明，它们经常由模块单位组成。这种组织提供了通过重新组合领域来创造新功能的机会；事实上，这种现象在自然界和生物技术实验室中都存在。然而，使用LacI/GalR家族转录调节因子，从分离的或在其他完整蛋白质的研究中，不能可靠地预测给定结构域在新的背景下的功能。我们正在研究并开始了解单个DNA结合域的功能是如何改变的，当它正常连接的调节域被同源结构域取代时。这些同源二聚体蛋白的两个功能结构域并不直接相互接触。相反，相互作用是通过~18个氨基酸连接体介导的，它接触(1)DNA结合域，(2)DNA配体，(3)配对单体的连接体，和(4)调节域的一个表面。这些界面的变化可能是在新蛋白质的背景下改变结构域功能的一种机制。我们设计并构建了新的转录抑制因子，包括LacI的DNA结合域/连接区和其他大肠杆菌同源物的调节域。这种设计修改了连接子和调节域之间的接口。我们首先确定了蛋白质功能的哪些特征会被这个过程改变：初步结果表明，DNA亲和力、DNA特异性和变构可以在不同的调节域的存在下受到不同的影响。接下来，我们将确定连接子和调控结构域的哪些特定残基赋予这些独特的功能方面。我们将把这些结果与几个关于残基预测相一致，这些残基赋予单个成员独特的功能

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Sequence and structure analysis of proteins reveals that they are frequently comprised of modular units. Such organization provides opportunity to create novel functions by recombining domains; indeed, this phenomenon occurs in both nature and the biotech laboratory. However, the function of a given domain in a new context cannot be reliably predicted from studies of the domain in isolation or in the context of other intact proteins, using the LacI/GalR family of transcription regulators. We are studying and beginning to understand how the function of a single DNA-binding domain is altered when the regulatory domain to which it is joined normally is replaced with homologous domains. The two functional domains of these homodimeric proteins do not directly contact each other. Instead, interactions are mediated through an ~18 amino acid linker, which contacts (1) the DNA-binding domain, (2) DNA ligand, (3) the linker of the partner monomer, and (4) one surface of the regulatory domain. Changes in these interfaces may be one mechanism by which domain function is altered in the context of a new protein. We have designed and constructed novel transcription repressors comprising the DNA-binding domain/linker of LacI and the regulatory domains of other E. coli homologues. This design modifies the interface between the linker and the regulatory domain. We are first determining which features of protein function are altered by this process: Preliminary results show that DNA-affinity, DNA-specificity, and allostery can be differentially affected in the presence of different regulatory domains. We will next determine which specific residues of the linker and of the regulatory domains impart these unique aspects of function. We will reconcile these results with several predictions about residues that impart unique function to individual memb