Intrinsic curvature induced packing heterogeneity and non-uniform distribution of cholesterol and Abeta peptide in lipid bilayers

固有曲率诱导脂质双层中胆固醇和 Abeta 肽的堆积异质性和不均匀分布

• 批准号: 10656172
• 负责人: Radha Ranganathan
• 金额: $ 10.88万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10656172
• 关键词: Amyloid beta-Protein; Area; Behavior; Binding; Biological; Brain; Calibration; Characteristics; Charge; Cholesterol; Detection; Development; Electron Spin Resonance Spectroscopy; Electrostatics; Evolution; Exhibits; Fluorescence; Gel; Glycerol; Goals; Growth; Heterogeneity; Hydrophobicity; Individual; Knowledge; Length; Lipid Bilayers; Lipids; Liquid substance; Measures; Membrane; Membrane Fluidity; Methodology; Methods; Microscopy; Movement; Peptides; Pharmaceutical Preparations; Phase; Phosphorylcholine; Positioning Attribute; Property; Reporting; Research; Resolution; Shapes; Stress; Structure; Variant; Vesicle; experimental study; fluorophore; improved; innovation; laurdan; melting; neurotoxic; neurotoxicity; novel; preference; redshift; second harmonic

Project Summary: Lipid intrinsic curvature is a fundamental physical feature that regulates membrane interactions. The goal is to elucidate intrinsic curvature induced effects on the growth of membrane curvature and packing heterogeneity upon mixing lipids of dissimilar curvature and the consequent distribution of cholesterol and the neurotoxic 25-35 segment of Aβ peptides in bilayers, using a novel steady state fluorescence method that utilizes the computed second harmonic (SH) of the zeroth order spectra to better resolve spectral lines that are not obvious in the zeroth harmonic. The polarity sensitive fluorophore Laurdan will be employed. Laurdan emission in bilayers is a composite of a line, referred to as blue, from the denser packed region at 432 nm and a red line from the less packed region starting at 440 nm in single component gel phases evolving to 490 nm upon melting or mixing with a higher curvature lipid. This path of structure evolution was a new finding. The present hypothesis is: Any added molecule of a dissimilar curvature enters the less packed region of the bilayer. A higher curvature molecule increases stress which is then relieved by growth in flat domains. Laurdan’s preference for flat regions together with growth in such domains contribute to an increase in Laurdan presence in flat domains. Fluorescence spectra register an increase in the area, Ab, of the blue line from flat domains and decrease in the red line area, Ar, along with a red-shift of the red peak and no shift in the blue peak. A lower curvature molecule decreases stress and the membrane tends toward a homogeneous curvature distribution resulting in an Ab decrease, Ar increase, blue shift of the red peak and no shift of the blue peak. Enhancement of heterogeneity in regional curvature due to dissimilarity of intrinsic curvature of mixing lipids and preferential distribution of cholesterol or Aβ peptide that manifest distinct difference in behaviors of areas and peak positions are predicted. Laurdan fluorescence spectra will be measured without and with cholesterol or Aβ (25-35) in mixed bilayers of (i and ii) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with (i) 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), and (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol (POPG) and (iii) brain lipids. SH of spectra will be computed to determine the number of elementary emissions. Spectra will be fitted to this number of log-normal functions according to developed methodology to resolve spectral lines. Area and peak position changes with mixture composition will be examined to determine packing heterogeneity in the bilayers without the transmembrane molecules and the type of distribution (uniform or non-uniform) of these molecules. Computing the SH of fluorescence spectra by modulating the wavenumber in analogy to field modulation in ESR spectroscopy is an innovative approach to bring superior definition to features missed in broad featureless in measured zeroth harmonics. The proposed research will significantly enhance current knowledge by elucidating the key role of intrinsic curvature in the mechanism by which the added molecule enters, accumulates preferentially, inhomogenously rigidifies the bilayer and changes its shape.

项目概述：脂质固有曲率是调节膜的基本物理特征 交互.我们的目标是阐明内在曲率引起的影响的增长膜曲率 以及不同曲率的脂质混合后的包装异质性和由此产生的 胆固醇和神经毒性的Aβ肽25-35段的双层，使用一种新的稳态荧光 一种利用零阶光谱的计算二次谐波（SH）来更好地解析光谱的方法， 在零次谐波中不明显的线。将采用极性敏感荧光团Laurdan。 双层中的劳登发射是来自432处的较致密填充区域的线（称为蓝色）的合成 在单组分凝胶相中，从440 nm处开始的较少填充区域的红线演变为 在熔融或与更高曲率的脂质混合时，这种结构演化的路径是一个新的发现。 目前的假设是：任何添加的不同曲率的分子都进入了 双层。更高曲率的分子增加应力，然后通过在平坦域中生长来缓解应力。劳登的 对平坦区域的偏好以及在这些区域的生长有助于增加劳登的存在 在平坦的区域。荧光光谱记录了来自平坦域的蓝线的面积Ab的增加， 红线面积Ar沿着减小，同时红峰发生红移，而蓝峰无位移。较低 曲率分子降低应力，膜趋于均匀曲率分布 导致Ab降低，Ar增加，红峰蓝移，蓝峰无位移。增强 由于混合脂层的内禀曲率不同， 胆固醇或Aβ肽的分布，在峰面积和峰位行为上表现出明显差异 是预测的。将在无胆固醇或Aβ（25 - 35）和有胆固醇或Aβ（25-35）的情况下测量Laurdan荧光光谱， （i和ii）1，2-二棕榈酰基-sn-甘油基-3-磷酸胆碱（DPPC）与（i）1-棕榈酰基-2-油酰基-sn- 甘油-3-磷酸胆碱（POPC），和（ii）1-棕榈酰-2-油酰-sn-甘油-3-磷酸-（1′-rac-甘油（POPG） 和（iii）脑脂质。将计算光谱的SH以确定基元发射的数量。光谱 将根据所开发的方法来拟合对数正态函数的数量以解析谱线。 将检查面积和峰位置随混合物组成的变化，以确定填充异质性 在没有跨膜分子的双层中， 这些分子。模拟场调制波数计算荧光光谱的SH ESR光谱中的调制是一种创新的方法，可以将上级清晰度带到 在测量的零次谐波中没有广泛的特征。这项研究将大大提高目前 通过阐明内在曲率在添加分子的机制中的关键作用， 进入，优先聚集，不均匀地硬化双层并改变其形状。