Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

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• 作者：Ming Chi Yang ; Hong-Hsiang Guan ; Jinn-Moon Yang ; Cheng-Neng Ko ; Ming-Yih Liu ; Yih-Hung Lin ; Yen-Chieh Huang ; Chun-Jung Chen ; Simon J. T. Mao
• 刊名：Crystal Growth & Design
• 出版年：2008
• 出版时间：December 3, 2008
• 年：2008
• 卷：8
• 期：12
• 页码：4268-4276
• 全文大小：472K
• 年卷期：v.8,no.12(December 3, 2008)
• ISSN：1528-7505

文摘

β-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D₃ binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D₃ to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was “disrupted” by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D₃ in the complex by altering the pH and initial ratios of vitamin D₃/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D₃/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D₃ in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.