Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes.
Synthesis and Evaluation of a Library of Fluorescent Dipeptidomimetic Analogues as Substrates for Modified Bacterial Ribosomes.
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DOI：
10.1021/acs.biochem.6b00102
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发表时间：
2016-05-03
期刊：
影响因子：
2.9
通讯作者：
Hecht SM
中科院分区：
文献类型：
--
作者：
Chowdhury SR;Chauhan PS;Dedkova LM;Bai X;Chen S;Talukder P;Hecht SM
本文描述了基于芳基取代的恶唑和噻唑的二肽模拟物类似物文库的合成和生物物理表征，以及它们代替天然荧光团掺入绿色荧光蛋白（GFP）的66位。这些荧光类似物类似于由GFP天然形成的荧光团。正如预期的那样，类似物的物理化学性质随着苯环帕拉取代基的变化而变化。库中化合物的荧光发射波长最大值在约365 nm（近紫外区）至约490 nm（可见光区）之间变化。这些化合物还表现出较大范围的量子产率（0.01-0.92）。类似物被用来激活一个抑制tRNACUA，并纳入到位置66的GFP使用体外蛋白质生物合成系统，采用工程核糖体选择其能力，以纳入二肽。选择了四个具有有趣的物理性质和合理的抑制产率的类似物，并在更大规模上制备了含有这些荧光团的荧光蛋白（FPs）进行更详细的研究。当将FP与相应的氨酰-tRNA和实际的二肽类似物进行比较时，FP在相同浓度下表现出显著增强的荧光强度。这部分是由于存在的荧光团作为蛋白质骨架的内在元素。也有特征的变化，在发射最大值，表明这些探针的环境敏感性。吖啶酮-2-基丙氨酸和恶唑1a分别掺入GFP的39和66位，并显示形成有效的Förster共振能量转移（FRET）对，表明类似物可用作FRET探针。
Described herein are the synthesis and photophysical characterization of a library of aryl-substituted oxazole- and thiazole-based dipeptidomimetic analogues, and their incorporation into position 66 of green fluorescent protein (GFP) in lieu of the natural fluorophore. These fluorescent analogues resemble the fluorophore formed naturally by GFP. As anticipated, the photophysical properties of the analogues varied as a function of the substituents at the para position of the phenyl ring. The fluorescence emission wavelength maxima of compounds in the library varied from ~365 nm (near-UV region) to ~490 nm (visible region). The compounds also exhibited a large range of quantum yields (0.01–0.92). The analogues were used to activate a suppressor tRNACUA and were incorporated into position 66 of GFP using an in vitro protein biosynthesizing system that employed engineered ribosomes selected for their ability to incorporate dipeptides. Four analogues with interesting photophysical properties and reasonable suppression yields were chosen, and the fluorescent proteins (FPs) containing these fluorophores were prepared on a larger scale for more detailed study. When the FPs were compared with the respective aminoacyl-tRNAs and the actual dipeptide analogues, the FPs exhibited significantly enhanced fluorescence intensities at the same concentrations. Part of this was shown to be due to the presence of the fluorophores as an intrinsic element of the protein backbone. There were also characteristic shifts in the emission maxima, indicating the environmental sensitivity of these probes. Acridon-2-ylalanine and oxazole 1a were incorporated into positions 39 and 66 of GFP, respectively, and were shown to form an efficient Förster resonance energy transfer (FRET) pair, demonstrating that the analogues can be used as FRET probes.
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影响因子：
15
作者：
Maini R;Dedkova LM;Paul R;Madathil MM;Chowdhury SR;Chen S;Hecht SM
通讯作者：
Hecht SM
影响因子：
2.7
作者：
Roy Chowdhury S;Maini R;Dedkova LM;Hecht SM
通讯作者：
Hecht SM
影响因子：
3.6
作者：
Meyers, AI;Tavares, FX
通讯作者：
Tavares, FX
影响因子：
3.6
作者：
Sanz-Cervera, Juan F.;Blasco, Rauel;Fustero, Santos
通讯作者：
Fustero, Santos
影响因子：
14.9
作者：
ROBERTSON, SA;NOREN, CJ;SCHULTZ, PG
通讯作者：
SCHULTZ, PG