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Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Mar 1;66(Pt 3):364-72. doi: 10.1107/S1744309110002022. Epub 2010 Feb 27.

Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals.

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  • 1Case Western Reserve University, Department of Physiology and Biophysics, The Protein Expression Purification Crystallization Core, 10900 Euclid Avenue, Robbins Building E549, Cleveland, OH 44106-4970, USA.


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 approximately 84% of their surface area buried) and to be surrounded by partially polar microenvironments (with approximately 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 (approximately 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.

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