ALLOSTERIC DETERMINANTS IN THE LACI/GALR FAMILY

LACI/GALR 家族中的变构决定因素

• 批准号: 7381960
• 负责人: LISKIN SWINT-KRUSE
• 金额: $ 13.13万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381960
• 关键词: ALLOSTERIC; DETERMINANTS; LACI; GALR; FAMILY

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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。蛋白质的序列和结构分析表明，它们通常由模块化单元组成。这样的组织提供了通过重组域来创建新功能的机会。确实，这种现象在自然界和生物技术实验室都发生。但是，使用转录调节剂的LACI/GALR家族，不能从隔离或其他完整蛋白质中对域的研究或其他完整蛋白质的研究可靠地预测给定域的功能。我们正在研究并开始理解当通常将其连接到的调节域通常用同源域取代时，如何改变单个DNA结合域的功能。这些同二聚体蛋白的两个功能结构域不会直接接触。取而代之的是，相互作用是通过约18个氨基酸接头介导的，该氨基酸连接器接触（1）DNA结合域，（2）DNA配体，（3）伴侣单体的接头，以及（4）调节域的一个表面。这些接口的变化可能是一种机制，在新蛋白质的背景下，域功能会改变。我们已经设计和构建了新型转录阻遏物，其中包括LACI的DNA结合域/连接器以及其他大肠杆菌同源物的调节域。该设计修改了链接器与调节域之间的接口。我们首先确定蛋白质功能的哪些特征是通过此过程改变的：初步结果表明，在存在不同调节域的存在下，DNA亲和力，DNA特异性和变构可以受到差异影响。接下来，我们将确定链接器的哪些特定残基和调节域的赋予这些独特的功能方面。我们将通过几个关于残基的预测来调和这些结果