Broughton David P, Holod Chloe G, Camilo-Contreras Angelica, Harris Darcy R, Brewer Scott H, Phillips-Piro Christine M
Department of Chemistry, Franklin & Marshall College P.O. Box 3003 Lancaster PA 17604-3003 USA
RSC Adv. 2024 Oct 17;14(44):32284-32291. doi: 10.1039/d4ra05058d. eCollection 2024 Oct 9.
The photophysical properties of the β-barrel superfolder green fluorescent protein (sfGFP) arise from the chromophore that forms post-translationally in the interior of the protein. Specifically, the protonation state of the side chain of tyrosine 66 in the chromophore, in addition to the network of hydrogen bonds between the chromophore and surrounding residues, is directly related to the electronic absorbance and emission properties of the protein. The pH dependence of the photophysical properties of this protein were modulated by the genetic, site-specific incorporation of 3-nitro-l-tyrosine (mNOY) at site 66 in sfGFP. The altered photophysical properties of this noncanonical amino acid (ncAA) sfGFP construct were assessed by absorbance and fluorescence spectroscopies. Notably, a comparison of the p of the 3-nitrophenol side chain of mNOY incorporated in the protein relative to the phenol side chain of the tyrosine at site 66 in the native chromophore as well as the p of the 3-nitrophenol side chain of the free ncAA were measured and are compared. A structural analysis of the ncAA containing sfGFP construct is presented to yield molecular insight into the origin of the altered absorbance and fluorescence properties of the protein.
β桶超折叠绿色荧光蛋白（sfGFP）的光物理性质源于在蛋白质内部翻译后形成的发色团。具体而言，发色团中酪氨酸66侧链的质子化状态，以及发色团与周围残基之间的氢键网络，直接关系到蛋白质的电子吸收和发射特性。通过在sfGFP的66位点进行遗传、位点特异性掺入3-硝基-L-酪氨酸（mNOY），调节了该蛋白质光物理性质的pH依赖性。通过吸收光谱和荧光光谱评估了这种非天然氨基酸（ncAA）sfGFP构建体改变后的光物理性质。值得注意的是，测量并比较了蛋白质中掺入的mNOY的3-硝基苯酚侧链相对于天然发色团中66位点酪氨酸的苯酚侧链的pKa，以及游离ncAA的3-硝基苯酚侧链的pKa。对含ncAA的sfGFP构建体进行了结构分析，以深入了解蛋白质吸收和荧光性质改变的起源。
