Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.
Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.
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DOI：
10.1021/acs.biochem.0c00474
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发表时间：
2020-09-22
期刊：
影响因子：
2.9
通讯作者：
Mills JH
中科院分区：
文献类型：
--
作者：
Henderson JN;Simmons CR;Fahmi NE;Jeffs JW;Borges CR;Mills JH
基因编码荧光非规范氨基酸（fNCAAs）可用于开发新型的蛋白质功能荧光传感器。由于缺乏对含有fNCAAs的基于蛋白质的传感器的广泛的物理化学和结构表征，以前实现这一目标的努力受到限制。本文报道了结合人CD40L的5c8抗体中含有fncaa的Fab片段的稳态光谱特性和首次结构分析。先前报道的5c8变体中，轻链残基IleL98被fNCAA L-（7-羟基香豆素-4-基）乙基甘氨酸（7-HCAA）取代，在抗原结合后显示出1.7倍的荧光增加。对未结合和结合形式的7-HCAA表观pKa的测定和比较表明，观察到的荧光增加不是pKa扰动的结果。载脂蛋白和结合形式的含有fncaa的Fab的晶体结构揭示了7-HCAA侧链与抗原结合时被破坏的周围残基之间的相互作用。这种结构表征不仅提供了对蛋白质环境如何调节7-HCAA荧光特性的深入了解，而且可以作为合理设计新的基于荧光蛋白的蛋白质功能报告者的起点。
Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue IleL98 is replaced with the fNCAA L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent pKas of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in pKa. Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.
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影响因子：
2.3
作者：
PINES, E;FLEMING, GR
通讯作者：
FLEMING, GR
影响因子：
16.6
作者：
Lacey, Vanessa K.;Parrish, Angela R.;Han, Shuliang;Shen, Zhouxin;Briggs, Steven P.;Ma, Yuguo;Wang, Lei
通讯作者：
Wang, Lei
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1126/science.aaa2424
发表时间：
2015-02-20
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Pearson AD;Mills JH;Song Y;Nasertorabi F;Han GW;Baker D;Stevens RC;Schultz PG
通讯作者：
Schultz PG
影响因子：
4.4
作者：
PINES, E;HUPPERT, D;AGMON, N
通讯作者：
AGMON, N