UV, CD, and NMR studies of Amphotericin B in nanoscale discoidal lipid bilayers

纳米级盘状脂质双层中两性霉素 B 的 UV、CD 和 NMR 研究

• 批准号: 8436306
• 负责人: Thomas Michael Anderson
• 金额: $ 4.69万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-16 至 2014-03-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8436306
• 关键词: Affinity; Amphotericin B; Antifungal Agents; Binding; Carbon; Cell membrane; Chemicals; Cholesterol; Complex; Dependence; Development; Dimerization; Effectiveness; Environment; Ergosterol; Health; High Pressure Liquid Chromatography; Human; Ion Channel; Kidney; Length; Lighting; Lipid Bilayers; Lipids; Liposomes; Measures; Membrane; Membrane Proteins; Molecular; Monitor; Nephrotoxic; Pharmacologic Substance; Preparation; Research; Resolution; Specificity; Sterols; Structural Protein; Structure; System; Tail; Therapeutic Index; Toxic effect; Yeasts; base; design; hemiketal; improved; intermolecular interaction; membrane model; mycosamine; nanodisk; nanoscale; physical state; preference; protein structure; research study; solid state nuclear magnetic resonance; stoichiometry; success

DESCRIPTION (provided by applicant): The clinically vital, but severely nephrotoxic, antifungal Amphotericin B has a unique mechanism of action; rather than bind to a macromolecular target, it self-assembles into membrane ion channels in yeast membranes. The effectiveness of AmB arises from its affinity for ergosterol in yeast membranes. However, AmB is severely nephrotoxic due to a competing affinity for cholesterol in human cell membranes. Lack of a suitable model membrane for studying AmB has resulted directly in a lack of detailed molecular understanding of its sterol specificity, thus severely limiting the design of more effective and /or less nephrotoxic derivatives. The typical model membrane employed is the liposome, however, AmB forms extremely large "hyper-aggregates" in liposomes. Interestingly, analysis of membrane protein structure has also been limited by tendency for aggregation and poor model membranes. Recently, nanoscale discoidal lipid bilayers (nanodiscs) have proven to be effective model membranes for studying structure of monomeric membrane proteins. We propose to harness the advantages of the nanodisc to analyze differences in structure and stoichiometry of the AmB/cholesterol and AmB/ergosterol channels. Quantitative analysis and UV and CD spectroscopic analysis will determine the AmB/nanodisc ratio in the presence of each sterol. UV and CD spectra of AmB will serve as a probe of the physical state of the incorporated AmB. Solid state NMR experiments (SSNMR) will be used to determine the channel length preference for cholesterol and ergosterol. ISCSIP rotational echo double resonance SSNMR will identify those AmB carbon atoms interacting with P atoms of the lipid. Finally, SSNMR analysis of AmB in the nanodisc will allow determination of specific atoms involved in AmB/ergosterol and AmB/cholesterol binding, as measured by changes in 13C chemical shift of the AmB carbons upon binding the sterol. Collectively, these studies will illuminate differences in the cholesterol and ergosterol AmB channel complexes which will serve as a starting point for the rational design of more effective, less nephrotoxic AmB derivatives.

描述（由申请人提供）：临床上至关重要，但严重肾毒性的抗真菌两性霉素B具有独特的作用机制；它不是与大分子靶标结合，而是自组装成酵母膜中的膜离子通道。AmB的有效性源于其对酵母膜麦角甾醇的亲和力。然而，由于AmB对人细胞膜中的胆固醇具有竞争性亲和力，因此具有严重的肾毒性。缺乏合适的模型膜来研究AmB，直接导致对其甾醇特异性缺乏详细的分子理解，从而严重限制了更有效和/或更少肾毒性衍生物的设计。典型的膜模型是脂质体，然而，AmB在脂质体中形成极大的“超聚集体”。有趣的是，膜蛋白结构的分析也受到聚集倾向和不良模型膜的限制。近年来，纳米盘状脂质双层被证明是研究膜蛋白单体结构的有效模型膜。我们建议利用纳米盘的优势来分析AmB/胆固醇和AmB/麦角甾醇通道的结构和化学计量差异。定量分析以及紫外和CD光谱分析将确定每种甾醇存在时AmB/纳米盘的比例。AmB的紫外和CD光谱将作为掺入的AmB的物理状态的探针。固体核磁共振实验（SSNMR）将用于确定通道长度对胆固醇和麦角甾醇的偏好。ISCSIP旋转回声双共振SSNMR将识别与脂质P原子相互作用的AmB碳原子。最后，纳米圆盘中AmB的ssmr分析将允许确定参与AmB/麦角甾醇和AmB/胆固醇结合的特定原子，通过测量与甾醇结合时AmB碳的13C化学位移的变化。总的来说，这些研究将阐明胆固醇和麦角甾醇AmB通道复合物的差异，这将作为合理设计更有效、肾毒性更小的AmB衍生物的起点。