Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland 21212, USA.
Protein Sci. 2013 Apr;22(4):475-85. doi: 10.1002/pro.2234. Epub 2013 Mar 8.
The ability to regulate cellular protein activity offers a broad range of biotechnological and biomedical applications. Such protein regulation can be achieved by modulating the specific protein activity or through processes that regulate the amount of protein in the cell. We have previously demonstrated that the nonhomologous recombination of the genes encoding maltose binding protein (MBP) and TEM1 β-lactamase (BLA) can result in genes that confer maltose-dependent resistance to β-lactam antibiotics even though the encoded proteins are not allosteric enzymes. We showed that these phenotypic switches-named based on their conferral of a switching phenotype to cells-resulted from a specific interaction with maltose in the cell that increased the switches cellular accumulation. Since phenotypic switches represent an important class of engineered proteins for basic science and biotechnological applications in vivo, we sought to elucidate the phenomena behind the increased accumulation and switching properties. Here, we demonstrate the key role for the linker region between the two proteins. Experimental evidence supports the hypothesis that in the absence of their effector, some phenotypic switches possess an increased rate of unfolding, decreased conformational stability, and increased protease susceptibility. These factors alone or in combination serve to decrease cellular accumulation. The effector functions to increase cellular accumulation by alleviating one or more of these defects. This perspective on the mechanism for phenotypic switching will aid the development of design rules for switch construction for applications and inform the study of the regulatory mechanisms of natural cellular proteins.
调节细胞内蛋白质活性的能力为生物技术和生物医学应用提供了广泛的应用前景。这种蛋白质调节可以通过调节特定蛋白质的活性或通过调节细胞内蛋白质含量的过程来实现。我们之前已经证明，编码麦芽糖结合蛋白（MBP）和 TEM1 β-内酰胺酶（BLA）的基因的非同源重组可以导致基因赋予麦芽糖依赖性对β-内酰胺抗生素的抗性，尽管编码的蛋白质不是变构酶。我们表明，这些表型开关-根据它们赋予细胞开关表型而命名-是由于细胞内麦芽糖的特异性相互作用导致的，这增加了开关的细胞积累。由于表型开关是基础科学和体内生物技术应用的一类重要的工程蛋白，我们试图阐明增加积累和开关特性背后的现象。在这里，我们证明了两个蛋白质之间的连接区的关键作用。实验证据支持这样的假设，即没有它们的效应物时，一些表型开关具有更高的解折叠速率、降低的构象稳定性和增加的蛋白酶敏感性。这些因素单独或组合作用会降低细胞积累。效应物通过缓解这些缺陷中的一个或多个来增加细胞积累。这种对表型开关机制的看法将有助于为开关构建的设计规则的发展提供信息，为自然细胞蛋白质的调控机制研究提供信息。
