Rational design for crystallization of β-lactoglobulin and vitamin D3 complex: Revealing a secondary binding site

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

*Corresponding author for this work

研究成果: Article同行評審

23 引文 斯高帕斯(Scopus)

摘要

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 D 3 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.

原文English
頁(從 - 到)4268-4276
頁數9
期刊Crystal Growth and Design
8
發行號12
DOIs
出版狀態Published - 1 十二月 2008

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