Structural Biology and Crystallization Communications Evaluating the Efficacy of Tryptophan Fluorescence and Absorbance as a Selection Tool for Identifying Protein Crystals
Structural Biology and Crystallization Communications Evaluating the Efficacy of Tryptophan Fluorescence and Absorbance as a Selection Tool for Identifying Protein Crystals
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环境中的个人Dahans在已知的蛋白质结构和四个商业机器人UV显微镜照亮Dahans含蛋白质晶体的色氨酸荧光（落射照明）或吸光度（传输）的有效性进行了评估。与其他研究一致，色氨酸残基被发现平均大部分被埋在蛋白质结构中（其表面积的84%被埋），并被部分极性微环境包围（其表面积的43%被极性残基覆盖），这表明荧光信号猝灭的固有程度。在细菌基因组中，多达三分之一（平均18.5%）的开放阅读框架缺乏色氨酸。在实验室中，由于坐滴和悬滴结晶试验中常用的介质会衰减紫外光，因此通常需要通过手动移除或倒置支持介质来简化光路。长时间暴露于UV光（分钟）会使一些蛋白质样品沉淀。介绍了许多市售介质在结晶试验中的吸收光谱。讨论了用色氨酸吸光度表征晶体的优点。
The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with $84% of their surface area buried) and to be surrounded by partially polar microenvironments (with $43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third ($18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed.