Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.
Donor-Acceptor Pyridinium Salts for Photo-Induced Electron-Transfer-Driven Modification of Tryptophan in Peptides, Proteins, and Proteomes Using Visible Light.
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DOI：
10.1021/jacs.1c10536
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发表时间：
2022-04-13
影响因子：
15
通讯作者：
Taylor, Michael T.
中科院分区：
文献类型：
--
作者：
Hoopes, Caleb R.;Garcia, Francisco J.;Sarkar, Akash M.;Kuehl, Nicholas J.;Barkan, David T.;Collins, Nicole L.;Meister, Glenna E.;Bramhall, Taylor R.;Hsu, Chien-Hsiang;Jones, Michael D.;Schirle, Markus;Taylor, Michael T.
色氨酸（Trp）在蛋白质生物化学中发挥着多种关键的功能作用，然而，由于其低固有频率和较差的亲核性，设计有效的色氨酸单蛋白生物偶联和原位化学蛋白质组学分析方法仍然是一个挑战。在这里，我们报告了一种共价色氨酸修饰方法，该方法通过调用光诱导电子转移（PET）作为驱动高效反应性的手段，适用于这两种情况。我们设计了联芳基n -氨基甲酰吡啶盐，它具有供体-受体关系，可以用可见光触发光学，同时衰减探针的光氧化电位，以防止光降解。该探针在8个肽和蛋白质的小库中进行了检测，发现微摩尔浓度的探针和短时间的紫光照射（10-60分钟）对表面暴露的色氨酸残基具有有效的反应性。氨基甲酸酯转移基团可用于将有用的官能团转移到蛋白质上，包括亲和标签和点击手柄。DFT计算和其他机制分析揭示了激发态寿命、相对荧光量子产率和化学反应性之间的相关性。利用生物素化和叠氮化吡啶盐对HEK293T裂解物和HEK293T细胞进行色氨酸谱分析，采用440 nm LED照射。活细胞标记实验的肽水平富集鉴定出290个Trp修饰，与其他π-氨基酸相比，Trp修饰选择性为82%；证明了该方法鉴定和量化活细胞活性色氨酸残基的能力。
Tryptophan (Trp) plays a variety of critical functional roles in protein biochemistry however, owing to its low natural frequency and poor nucleophilicity, the design of effective methods for both single protein bioconjugation at Trp as well as for in situ chemoproteomic profiling remains a challenge. Here, we report a method for covalent Trp modification that is suitable for both scenarios by invoking photo-induced electron transfer (PET) as a means of driving efficient reactivity. We have engineered biaryl N-carbamoyl pyridinium salts that possess a donor-acceptor relationship which enables optical triggering with visible light whilst simultaneously attenuating the probe’s photo-oxidation potential in order to prevent photodegradation. This probe was assayed against a small bank of eight peptides and proteins, where it was found that micromolar concentrations of probe and short irradiation times (10–60 min) with violet light enabled efficient reactivity towards surface exposed Trp residues. The carbamate transferring group can be used to transfer useful functional groups to proteins including affinity tags and click handles. DFT calculations and other mechanistic analyses reveal correlations between excited state lifetimes, relative fluorescent quantum yields, and chemical reactivity. Biotinylated and azide-functionalized pyridinium salts were used for Trp profiling in HEK293T lysates and in situ in HEK293T cells using 440 nm LED irradiation. Peptide level enrichment from live cell labelling experiments identified 290 Trp modifications, with an 82% selectivity for Trp modification over other π-amino acids; demonstrating the ability of this method to identify and quantify reactive Trp residues from live cells.
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影响因子：
15
作者：
Antos JM;McFarland JM;Iavarone AT;Francis MB
通讯作者：
Francis MB
DOI：
10.1038/nrm.2017.53
发表时间：
2017-10
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Ray Chaudhuri A;Nussenzweig A
通讯作者：
Nussenzweig A
DOI：
10.1126/science.1216338
发表时间：
2012-05-11
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Langelier MF;Planck JL;Roy S;Pascal JM
通讯作者：
Pascal JM
影响因子：
46.2
作者：
Chan WC;Sharifzadeh S;Buhrlage SJ;Marto JA
通讯作者：
Marto JA
影响因子：
5.6
作者：
Barik S
通讯作者：
Barik S