School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China.
Biochem Biophys Res Commun. 2010 Jan 1;391(1):709-15. doi: 10.1016/j.bbrc.2009.11.125. Epub 2009 Nov 26.
The Methanococcus jannaschii tRNA(Tyr)/tyrosyl-tRNA synthetase pair has been engineered to incorporate unnatural amino acids into proteins in Escherichia coli site-specifically. In order to add other unnatural amino acids into proteins by this approach, the amino acid binding site of M. jannaschii tyrosyl-tRNA synthetase need to be mutated. The crystal structures of M. jannaschii tyrosyl-tRNA synthetase and its mutations were determined, which provided an opportunity to design aminoacyl-tRNA synthetases specific for other unnatural amino acids. In our study, we attempted to design aminoacyl-tRNA synthetases being able to deliver p-acetyl-L-phenylalanine into proteins. p-Acetyl-L-phenylalanine was superimposed on tyrosyl in M. jannaschii tyrosyl-tRNA synthetase-tyrosine complex. Tyr32 needed to be changed to non-polar amino acid with shorter side chain, Val, Leu, Ile, Gly or Ala, in order to reduce steric clash and provide hydrophobic environment to acetyl on p-acetyl-L-phenylalanine. Asp158 and Ile159 would be changed to specific amino acids for the same reason. So we designed 60 aminoacyl-tRNA synthetases. Binding of these aminoacyl-tRNA synthetases with p-acetyl-L-phenylalanine indicated that only 15 of them turned out to be able to bind p-acetyl-L-phenylalanine with reasonable poses. Binding affinity computation proved that the mutation of Tyr32Leu and Asp158Gly benefited p-acetyl-L-phenylalanine binding. And two of the designed aminoacyl-tRNA synthetases had considerable binding affinities. They seemed to be very promising to be able to incorporate p-acetyl-L-phenylalanine into proteins in E. coli. The results show that the combination of homology modeling and molecular docking is a feasible method to filter inappropriate mutations in molecular design and point out beneficial mutations.
产甲烷球菌 tRNA(Tyr)/酪氨酸-tRNA 合成酶对已被改造用于在大肠杆菌中定点将非天然氨基酸掺入蛋白质。为了通过这种方法将其他非天然氨基酸掺入蛋白质中，需要对产甲烷球菌酪氨酸-tRNA 合成酶的氨基酸结合位点进行突变。产甲烷球菌酪氨酸-tRNA 合成酶及其突变体的晶体结构已被确定，这为设计针对其他非天然氨基酸的氨酰-tRNA 合成酶提供了机会。在我们的研究中，我们试图设计能够将 p-乙酰-L-苯丙氨酸递送到蛋白质中的氨酰-tRNA 合成酶。将 p-乙酰-L-苯丙氨酸叠加在产甲烷球菌酪氨酸-tRNA 合成酶-酪氨酸复合物中的酪氨酸上。为了减少空间位阻并为 p-乙酰-L-苯丙氨酸上的乙酰基提供疏水环境，需要将 Tyr32 改变为具有较短侧链的非极性氨基酸，如 Val、Leu、Ile、Gly 或 Ala。出于同样的原因，Asp158 和 Ile159 也将被改变为特定的氨基酸。因此，我们设计了 60 种氨酰-tRNA 合成酶。这些氨酰-tRNA 合成酶与 p-乙酰-L-苯丙氨酸的结合表明，只有 15 种氨酰-tRNA 合成酶能够以合理的构象结合 p-乙酰-L-苯丙氨酸。结合亲和力计算证明，Tyr32Leu 和 Asp158Gly 的突变有利于 p-乙酰-L-苯丙氨酸的结合。并且两种设计的氨酰-tRNA 合成酶具有相当大的结合亲和力。它们似乎很有希望能够将 p-乙酰-L-苯丙氨酸掺入大肠杆菌中的蛋白质中。结果表明，同源建模和分子对接的结合是一种可行的方法，可以在分子设计中筛选出不合适的突变，并指出有益的突变。
