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Observation of a Partially-Opened Triple-Helix Conformation in 1 3-beta-Glucan by Fluorescence Resonance Energy Transfer Spectroscopy

Shih-Houng Young, Robert R. Jacobs and Wen-Ji Dong


 
ABSTRACT

Molecular conformation is one of the important factors in understanding the structure-activity relationship of 1 3-b-glucan. However, a direct observation of glucan conformation in solution is very difficult. Therefore, the fluorescence resonance energy transfer (FRET) spectroscopy was applied as an indirect method to study the conformational change of glucans induced by NaOH. Three conformations of glucan have been reported in solution: triple-helix, single helix, and random coil. Previous studies have suggested that treatment of the triple-helix conformation with NaOH produces single helix conformers. We propose that a partially-opened triple-helix rather than a single helix, is formed by treating the triple-helix glucan, laminarin with NaOH. Fluorescence resonance energy transfer (FRET) spectra of the glucan, laminarin, doubly labeled with 1-aminopyrene (AP) as donor probe and fluorescein-5-isothiocyanate (FITC) as acceptor probe attached at the reducing end, showed that a partially-opened triple-helix conformer was formed on treatment with NaOH. The conversion between the triple-helix and partially-opened conformation was reversible (when denaturation was mild, [NaOH] < 1 M). Different degrees of conformer opening could be stabilized by adding aniline blue, which binds to the single helix portion the partially-opened triple-helix glucan. Different conformations were stabilized with aniline blue when examined using the Limulus amoebocyte lysate (LAL). The results show that conformations stabilized at different times activate the LAL assay differently, thus demonstrating that the biological activity was dependent on the degree of strand opening.



Reprinted from Proceedings of the 1999 Beltwide Cotton Conferences pp. 180 - 184
©National Cotton Council, Memphis TN

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Document last modified Monday, Jun 21 1999