Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.
Steric and thermodynamic limits of design for the incorporation of large unnatural amino acids in aminoacyl-tRNA synthetase enzymes.
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DOI：
10.1002/prot.22706
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发表时间：
2010-06
影响因子：
2.9
通讯作者：
Brooks, Charles L., III
中科院分区：
文献类型：
--
作者：
Armen, Roger S.;Schiller, Stefan M.;Brooks, Charles L., III
来自古细菌的正交氨酰-tRNA合成酶/tRNA对已经进化为促进非天然氨基酸在大肠杆菌中的蛋白质中的位点特异性体内掺入。使用这种方法，非天然氨基酸已成功地掺入高翻译效率和保真度。在这项研究中，CHARMM为基础的分子对接和自由能计算用于评估氨酰-tRNA合成酶的特异性蛋白质-配体相互作用的合理设计。一系列新的非天然氨基酸配体对接到对苯甲酰基-L-苯丙氨酸tRNA合成酶，这表明，酶的结合口袋不提供足够的空间显着更大的配体。将特异性结合位点残基突变为丙氨酸以产生额外的空间以容纳更大的靶配体，然后引入突变以提高结合自由能。该方法用于重新设计几种不同靶配体的结合位点，然后针对标准20种氨基酸进行测试以验证靶特异性。预测只有设计用于结合Man-α-O-Tyr的合成酶对靶配体具有足够的选择性，并且也是药物稳定的。我们的研究表明，大体积配体的tRNA酶结合口袋的广泛重新设计可能是非常不利的。
Orthogonal aminoacyl-tRNA synthetase/tRNA pairs from archaea have been evolved to facilitate site specific in vivo incorporation of unnatural amino acids into proteins in Escherichia coli. Using this approach, unnatural amino acids have been successfully incorporated with high translational efficiency and fidelity. In this study, CHARMM-based molecular docking and free energy calculations were used to evaluate rational design of specific protein-ligand interactions for aminoacyl-tRNA synthetases. A series of novel unnatural amino acid ligands were docked into the p-benzoyl-L-phenylalanine tRNA synthetase, which revealed that the binding pocket of the enzyme does not provide sufficient space for significantly larger ligands. Specific binding site residues were mutated to alanine to create additional space to accommodate larger target ligands, and then mutations were introduced to improve binding free energy. This approach was used to redesign binding sites for several different target ligands, which were then tested against the standard 20 amino acids to verify target specificity. Only the synthetase designed to bind Man-α-O-Tyr was predicted to be sufficiently selective for the target ligand and also thermodynamically stable. Our study suggests that extensive redesign of the tRNA synthatase binding pocket for large bulky ligands may be quite thermodynamically unfavorable.
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