Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
Biochemistry. 2012 Jan 10;51(1):83-9. doi: 10.1021/bi2012542. Epub 2011 Dec 12.
Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.
两性霉素 B（AmB）被认为在麦角固醇存在下形成离子通道组装来发挥其抗真菌活性。在本研究中，我们旨在使用旋转回波双共振（REDOR）谱阐明两性霉素 B 和麦角固醇在水合磷脂双层中分子相互作用的模式。我们首先用 C14-（19）F 标记的两性霉素 B 和生物合成（13）C 标记的麦角固醇进行（13）C{（19）F}REDOR 实验，并暗示两性霉素 B-麦角固醇相互作用在双层中存在“头对头”和“头对尾”两种取向。为了进一步证实“头对尾”配对，合成了二甲基末端（C26/C27）标记的（13）C 标记的麦角固醇，并进行了 REDOR 测量。该光谱明确证明了两性霉素 B-麦角固醇配对存在“头对尾”取向。为了获得麦角固醇在膜中二甲末端位置的信息，使用标记的麦角固醇和 POPC 头部的磷原子进行了（13）C{（31）P}REDOR。在存在两性霉素 B 的情况下，麦角固醇的 C26/C27 信号处观察到明显的 REDOR 去相位，但在不存在两性霉素 B 的情况下则没有，这清楚地表明两性霉素 B 复合物中麦角固醇的侧链末端靠近双层表面。
