ALLOSTERIC DETERMINANTS IN THE LACI/GALR FAMILY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 蛋白质的序列和结构分析表明，它们通常由模块单元组成。 这样的组织提供了通过重组结构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，在一个新的背景下，一个给定的域的功能不能可靠地预测从研究的结构域在隔离或在其他完整的蛋白质的情况下，使用的LacI/GalR家族的转录调节。 我们正在研究并开始了解当一个DNA结合域的调节域被同源域取代时，它的功能是如何改变的。 这些同源二聚体蛋白质的两个功能结构域不直接彼此接触。 相反，相互作用是通过一个约18个氨基酸的接头介导的，该接头接触（1）DNA结合结构域，（2）DNA配体，（3）配偶体单体的接头，和（4）调节结构域的一个表面。 这些界面的变化可能是结构域功能在新蛋白质中改变的一种机制。 我们设计并构建了新的转录抑制子，包括LacI的DNA结合结构域/接头和其他大肠杆菌的调节结构域。coli同源物。 该设计修饰接头和调节结构域之间的界面。 我们首先确定蛋白质功能的哪些特征被这一过程改变：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结构域的存在下受到不同的影响。 接下来我们将确定接头和调节结构域的哪些特定残基赋予这些独特的功能。 我们将调和这些结果与几个预测残基赋予独特的功能，以个别成员。

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 members.