Genetic incorporation of D-amino acids into green fluorescent protein based on polysubstrate specificity
Genetic incorporation of D-amino acids into green fluorescent protein based on polysubstrate specificity
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基于多底物特异性将 D-氨基酸基因整合到绿色荧光蛋白中
DOI：
10.1039/c5ra02289d
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发表时间：
2015-01-01
期刊：
影响因子：
3.9
通讯作者：
Chen, Yijun
中科院分区：
文献类型：
--
作者：
Ma, Hairong;Liu, Nan;Chen, Yijun
D-氨基酸广泛存在于生物体中，在蛋白质折叠和功能中发挥重要作用。通过蛋白质合成机制基因整合D-氨基酸一直是蛋白质工程中的探索性任务，并且非常诱人但困难。在目前的工作中，一些D-氨基酸的基因掺入到绿色荧光蛋白使用多底物特异性的tRNA合成酶与同源的tRNA在大肠杆菌，和GFPuv突变体含有D-苯丙氨酸的荧光基团在残基66的特点。在位置66处的苯丙氨酸的立体化学切换导致发射和激发最大值的红移，并且显著提高了蛋白质的热稳定性。分子模拟进一步揭示，Phe66在GFPuv中的相反构型产生了两种各自的异构荧光团，其表现出独特的光谱特性和热稳定性。本研究通过体内核糖体翻译扩展蛋白质分子的骨架立体化学，以促进蛋白质工程。
D-amino acids are widely distributed in living organisms and are suggested to play important roles in protein folding and function. Genetic incorporation of D-amino acids through protein synthesis machinery has been an exploratory task in protein engineering, and is greatly enticing but difficult. In the present work, a number of D-amino acids were genetically incorporated into green fluorescent protein using a polysubstrate-specific tRNA synthetase with cognate tRNA in Escherichia coli, and the GFPuv mutant containing D-phenylalanine in the fluorophore at residue 66 was characterized. Stereochemical switching of phenylalanine at position 66 resulted in red shifts in the emission and excitation maxima and significantly improved the thermal stability of the protein. Molecular modeling further revealed that the opposite configurations of Phe66 in GFPuv produced two respective isomeric fluorophores that exhibit distinctive spectral properties and thermal stability. The present study expands the backbone stereochemistry of protein molecules by in vivo ribosomal translation to facilitate protein engineering.