Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment.
Structural Basis for Blocked Excited State Proton Transfer in a Fluorescent, Photoacidic Non-Canonical Amino Acid-Containing Antibody Fragment.
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DOI：
10.1016/j.jmb.2022.167455
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发表时间：
2022-04-30
影响因子：
5.6
通讯作者：
Mills, Jeremy H.
中科院分区：
文献类型：
--
作者：
Henderson, J. Nathan;Simmons, Chad R.;Mills, Jeremy H.
荧光非正则氨基酸L-(7-羟基香豆素-4-基)乙基甘氨酸(7-HCAA)含有光酸性的7-羟基香豆素(7-HC)侧链，其荧光性质可以通过环境调节。在蛋白质中，7-HCAA的荧光光谱发生了许多变化，包括强度的增加和减少以及红移和蓝移的最大发射。能够合理地设计蛋白质环境，以可预测的方式改变7-HCAA的荧光特性，可能会导致基于蛋白质的生物功能新型传感器的出现。然而，这些努力可能由于缺乏含有7-HCAA的蛋白质的结构特征而受到限制。在这里，我们报告了含有7-HCAA的抗体片段(以载脂蛋白和抗原结合的形式)的稳态光谱和X射线结晶学表征，其中观察到相对于游离氨基酸的基本上蓝移的7-HCAA发射最大值(~70 nm)。我们对这些蛋白质的结构表征提供了证据，表明蓝移是7-HC苯酚的激发态质子转移(ESPT)几乎完全被与蛋白质骨架相互作用阻止的结果。此外，抗原中的残基和荧光团之间的直接相互作用进一步阻止了相对于载脂蛋白的质子转移。这里报道的7-HCAA发射前所未有的蓝移的结构基础为开发新的基于荧光蛋白的传感器提供了一个框架。
The fluorescent non-canonical amino acid (fNCAA) L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) contains a photoacidic 7-hydroxycoumarin (7-HC) side chain whose fluorescence properties can be tuned by its environment. In proteins, many alterations to 7-HCAA’s fluorescence spectra have been reported including increases and decreases in intensity and red- and blue-shifted emission maxima. The ability to rationally design protein environments that alter 7-HCAA’s fluorescence properties in predictable ways could lead to novel protein-based sensors of biological function. However, these efforts are likely limited by a lack of structural characterization of 7-HCAA-containing proteins. Here, we report the steady-state spectroscopic and x-ray crystallographic characterization of a 7-HCAA-containing antibody fragment (in the apo and antigen-bound forms) in which a substantially blue-shifted 7-HCAA emission maximum (~70 nm) is observed relative to the free amino acid. Our structural characterization of these proteins provides evidence that the blue shift is a consequence of the fact that excited state proton transfer (ESPT) from the 7-HC phenol has been almost completely blocked by interactions with the protein backbone. Furthermore, a direct interaction between a residue in the antigen and the fluorophore served to further block proton transfer relative to the apoprotein. The structural basis of the unprecedented blue shift in 7-HCAA emission reported here provides a framework for the development of new fluorescent protein-based sensors.
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含有荧光非规范氨基酸的蛋白质中配体结合后光谱特性改变的结构起源。
影响因子：
2.9
作者：
Gleason PR;Kolbaba-Kartchner B;Henderson JN;Stahl EP;Simmons CR;Mills JH
通讯作者：
Mills JH
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
影响因子：
2.9
作者：
Henderson JN;Simmons CR;Fahmi NE;Jeffs JW;Borges CR;Mills JH
通讯作者：
Mills JH
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
影响因子：
8.4
作者：
Dumas A;Lercher L;Spicer CD;Davis BG
通讯作者：
Davis BG