Structural Studies of Bacterial Luciferase: Subunit Folding and Dimer Assembly in Vivo

一个新翻译的线性多肽如何折叠成功能性的天然蛋白质是当今蛋白质生物化学的核心问题。本研究的目的是描述细菌荧光素酶亚基在体内折叠和组装成功能性异二聚体酶的机制。两个亚基luxA和luxB的基因已被克隆并在大肠杆菌中表达。因此，亚基可以很容易地制备。此外，它们的结构可以很容易地通过寡核苷酸定向位点特异性突变改变。测量低光强度的便利性允许在6个数量级范围内检测突变体中的特定活性。因此，荧光素酶异二聚体为研究体内蛋白质折叠的原理提供了一个理想的系统。本项目将制备亚基的突变体，并通过折叠和组装过程的动力学和物理表征来分析它们结合形成活性酶的方式。

How a newly translated linear polypeptide folds into a functional native protein is the central issue in protein biochemistry today. The purpose of the proposed research is to delineate the mechanism by which the subunits of bacterial luciferase fold and assemble into the functional heterodimeric enzyme in vivo. Genes for the two subunits, luxA and luxB, have been cloned and expressed in E. coli. Thus, the subunits can be easily prepared. Moreover, their structures can be readily altered by oligonucleotide-directed site specific mutagenesis. The ease with which low light intensities can be measured permits the detection of specific activites in mutants over a range of six orders of magnitude. Thus, the luciferase heterodimer provides an ideal system for studying the principles which govern protein folding in vivo. In this project mutants of the subunits will be prepared, and the way they combine to form active enzyme will be analyzed through kinetic and physical characterization of the folding and assembly process.