Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Biochemistry. 2011 Sep 27;50(38):8221-5. doi: 10.1021/bi201037r. Epub 2011 Aug 31.
Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter's ligase ((W37V)LplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, (E20G/W37T)LplA, that ligates Pacific Blue as efficiently as (W37V)LplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.
色氨酸 37 这个关键残基的突变，能使大肠杆菌硫辛酸连接酶（LplA）的底物特异性扩大，使比硫辛酸大得多的非天然探针得以被识别。然而，这种方法对于阴离子底物却并不成功。一个例子是蓝色荧光染料 Pacific Blue，它与 7-羟基香豆素是等排体，但后者的连接酶（(W37V)LplA）或任何色氨酸 37 点突变体都不能识别它。在这里，我们报告了一个结构导向的、双残基筛选矩阵的结果，该矩阵发现了一个 LplA 双突变体 (E20G/W37T)LplA，它与 Pacific Blue 的连接效率与 (W37V)LplA 与 7-羟基香豆素的连接效率一样高。该 Pacific Blue 连接酶在活细胞内、细胞表面和酸性内体中对重组蛋白进行特异性标记的用途得到了证明。
