Dippel Andrew B, Olenginski Gregory M, Maurici Nicole, Liskov Melanie T, Brewer Scott H, Phillips-Piro Christine M
Department of Chemistry, Franklin and Marshall College, PO Box 3003, Lancaster, PA 17604-3003, USA.
Acta Crystallogr D Struct Biol. 2016 Jan;72(Pt 1):121-30. doi: 10.1107/S2059798315022858. Epub 2016 Jan 1.
The X-ray crystal structures of superfolder green fluorescent protein (sfGFP) containing the spectroscopic reporter unnatural amino acids (UAAs) 4-cyano-L-phenylalanine (pCNF) or 4-ethynyl-L-phenylalanine (pCCF) at two unique sites in the protein have been determined. These UAAs were genetically incorporated into sfGFP in a solvent-exposed loop region and/or a partially buried site on the β-barrel of the protein. The crystal structures containing the UAAs at these two sites permit the structural implications of UAA incorporation for the native protein structure to be assessed with high resolution and permit a direct correlation between the structure and spectroscopic data to be made. The structural implications were quantified by comparing the root-mean-square deviation (r.m.s.d.) between the crystal structure of wild-type sfGFP and the protein constructs containing either pCNF or pCCF in the local environment around the UAAs and in the overall protein structure. The results suggest that the selective placement of these spectroscopic reporter UAAs permits local protein environments to be studied in a relatively nonperturbative fashion with site-specificity.
已确定在蛋白质两个独特位点含有光谱报告非天然氨基酸（UAA）4-氰基-L-苯丙氨酸（pCNF）或4-乙炔基-L-苯丙氨酸（pCCF）的超折叠绿色荧光蛋白（sfGFP）的X射线晶体结构。这些非天然氨基酸通过基因方式掺入到sfGFP中，位于蛋白质溶剂暴露的环区域和/或β桶上的部分埋藏位点。在这两个位点含有非天然氨基酸的晶体结构使得能够以高分辨率评估非天然氨基酸掺入对天然蛋白质结构的影响，并能够直接建立结构与光谱数据之间的关联。通过比较野生型sfGFP的晶体结构与在非天然氨基酸周围局部环境以及整个蛋白质结构中含有pCNF或pCCF的蛋白质构建体之间的均方根偏差（r.m.s.d.），对结构影响进行了量化。结果表明，这些光谱报告非天然氨基酸的选择性定位允许以相对非干扰的方式位点特异性地研究局部蛋白质环境。
