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

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)4268-4276
Number of pages9
JournalCrystal Growth and Design
Volume8
Issue number12
DOIs
StatePublished - 1 Dec 2008

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