POWRE: Novel Spectroscopic Methods for Assessing Peptide-Lipid Interactions

9720606 Murphy有几个令人信服的医学和生物技术的原因促使开发新的方法来探测涉及自缔合肽和脂质双层的复杂系统的机制和动力学。 关键问题包括：有多少肽结合在一起形成一个孔？肽-肽结合发生在肽-脂质结合之前、同时还是之后？为了与脂质双层相互作用，肽必须处于特定的聚集状态吗？ 本研究计划提出研究开发两种新方法来探索这类问题的可行性。 研究目标1是确定静态光散射方法是否可以应用于评价包埋在膜中的肽的数量和缔合状态。 选择pH敏感的成孔肽GALA和抗体-GALA衍生物与磷脂酰胆碱大单层囊泡混合作为本研究的模型系统。 标准的静态光散射方法产生关于溶液中颗粒的分子量和尺寸的信息，并且要求在感兴趣的样品和缓冲液两者上收集散射数据。 该方法将通过收集四组散射数据进行修改：单独的肽、单独的脂质体、单独的缓冲液和肽-脂质体混合物。 通过从肽-脂质体混合物的散射中依次减去单独的肽、单独的脂质体或单独的缓冲液的散射，可以推断脂质体中肽缔合的程度和每个脂质体的肽总数。 研究目的二是探讨利用表面等离子体共振（SPR）光谱技术研究肽结合状态对调节肽-脂结合的重要性的可行性。 本研究的模型系统将是β-淀粉样肽单体和与带负电荷的磷脂和磷脂-神经节苷脂混合物的单层缔合的原纤维。SPR最常用于测量配体与固定化受体的结合动力学。 在一种改进中，固定化受体可以被自组装定向脂质单层取代。 SPR的一个常见困难是缔合速率可能是质量传递，而不是动力学限制。 将研究将这一困难转化为资产的可行性。 由于原纤维具有比单体肽显著更慢的扩散率，因此质量传递速率的测量应指示与脂质层相关联的主要物质是单体的还是聚集的。 ***

9720606 Murphy There are several compelling medical and biotechnological reasons motivating the development of new methods to probe the mechanisms and kinetics of complex systems involving self-associating peptides and lipid bilayers. Key questions include: how many peptides associate together to form a pore? does peptide-peptide association occur prior to, coincident with, or subsequent to peptide-lipid association? must the peptide be in a specific aggregational state in order to interact with the lipid bilayer? This research plan proposes to investigate the feasibility of developing two new methods to probe these kinds of questions. Research objective 1 is to establish whether static light scattering methods can be applied to evaluate the number and association state of peptides embedded in membranes. The pH-sensitive pore-forming peptide GALA and an antibody-GALA derivative, mixed with phosphatidylcholine large unilamellar vesicles, was chosen as the model system for this investigation. Standard static light scattering methods yield information on molecular weight and size of particles in solution, and requires that scattering data be collected on both the sample of interest and the buffer. This approach will be modified by collecting four sets of scattering data: peptide alone, liposomes alone, buffer alone, and peptide-liposome mixture. By subtracting out in turn the scattering from peptide alone, liposomes alone, or buffer alone from the scattering of the peptide-liposome mixture, it may be possible to infer the extent of peptide association in the liposome and the total number of peptides per liposome. Research objective 2 is to investigate the feasibility of using surface plasmon resonance (SPR) spectroscopy to explore the importance of peptide association state in modulating peptide-lipid association. The model system for this investigation will be beta-amyloid peptide monomer and fibrils associating with monolayers of negatively-charged pho spholipids and phospholipid- ganglioside mixtures, SPR is used most frequently to measure binding kinetics of ligands to immobilized receptors. In a modification of this, the immobilized receptors can be replaced by a self-assembled oriented lipid monolayer. A commonly cited difficulty with SPR is that the rates of association can be mass transport, rather than kinetically, limited. The feasibility of turning this difficulty into an asset will be investigated. Since fibrils have significantly slower diffusivities than monomeric peptides, measurement of the mass transport rate should indicate whether the predominant species associating with the lipid layer is monomeric or aggregated. ***