The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors
The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors
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DOI：
10.1073/pnas.94.9.4366
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发表时间：
1997-04-29
影响因子：
11.1
通讯作者：
Hellinga, HW
中科院分区：
文献类型：
--
作者：
Marvin, JS;Corcoran, EE;Hellinga, HW
合理蛋白质设计是一种新兴的方法，用于测试结构和功能的一般理论，合理操纵功能的能力也提供了创造具有生物技术价值的新蛋白质的可能性，在这里，我们使用设计方法来测试目前对蛋白质中变构相互作用的结构原理的理解，并演示一个简单的变构系统如何形成一个通用结构的构建基础。生物传感器分子工程系统，我们已经确定了大肠杆菌麦芽糖结合蛋白中预测与其麦芽糖结合位点变构连接的区域，环境敏感的荧光团共价连接到这些区域内特定位点处由半胱氨酸突变引入的独特硫醇，如预测的那样，这种缀合物的荧光相对于麦芽糖结合协同变化，结合位点和报告基团的空间分离允许各自的内在性质被独立地操纵。只要保持变构连接，因此可以改变配体结合而不影响通过荧光的结合事件的转导。为了证明生物传感器技术的适用性，我们在麦芽糖结合位点引入了一系列点突变，降低了蛋白质对其配体的亲和力。这些突变体蛋白质已被组合在一个复合生物传感器能够测量底物浓度在5%的准确度在浓度范围跨越五个数量级。
Rational protein design is an emerging approach for testing general theories of structure and function, The ability to manipulate function rationally also offers the possibility of creating new proteins of biotechnological value, Here we use the design approach to test the current understanding of the structural principles of allosteric interactions in proteins and demonstrate how a simple allosteric system can form the basis for the construction of a generic biosensor molecular engineering system, We have identified regions in Escherichia coli maltose-binding protein that are predicted to be allosterically linked to its maltose-binding site, Environmentally sensitive fluorophores were covalently attached to unique thiols introduced by cysteine mutations at specific sites within these regions, The fluorescence of such conjugates changes cooperatively with respect to maltose binding, as predicted, Spatial separation of the binding site and reporter groups allows the intrinsic properties of each to be manipulated independently, Provided allosteric linkage is maintained, ligand binding can therefore be altered without affecting transduction of the binding event by fluorescence. To demonstrate applicability to biosensor technology, we hare introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins have been combined in a composite biosensor capable of measuring substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude.