EAGER: Interfacial disruption of supported lipid bilayers by invading peptides

这个急切的项目是“高风险和高回报”的，因为它涉及一种全新的方法来研究肽-脂界面相互作用的机制，该方法使用一种名为耗散石英晶体微天平(QCM-D)的自由振荡传感器。生命依赖于生物膜的完整性及其维持功能的能力。智力上的功绩。有目的地用多肽破坏细菌膜的完整性是杀灭有毒细菌的新策略。或者，保护哺乳动物细胞膜不受多肽或低聚物破坏是缓解阿尔茨海默病、S(和许多其他淀粉样蛋白相关疾病)(S)(AD)的一种策略。在这两种情况下，人们对多肽如何结合到生物膜(或被称为支持的脂双层，SLB的膜模拟物)以及它们如何聚集和穿透形成毛孔和/或诱导脂质损失知之甚少。相互作用的各种模型包括吸附在SLB表面的形成肽，在SLB释放和不释放脂类的情况下形成孔。以前，用界面张力(Langmuir-Blodgett(LB))、渗漏、光谱(核磁共振、电子顺磁共振、CD)和散射测量(光、X射线和中子反射法)等方法来分析蛋白质-脂质相互作用。局限性包括存在非自然的脂质-空气界面(LB)和低敏感性(核磁共振、EPR)。使用荧光或电流变化的泄漏测量对于确定SBL的邻接性是有用的，但不能提供太多的机械洞察力。散射技术是对建议的QCM-D测量的补充，将在橡树岭国家实验室(我们在2012年秋季进行中子反射测量的建议是成功的)继续研究这里描述的相同问题。这是一个复杂的界面结合、破坏和传输挑战，发生在SLB的表面。PI提出使用一种新的技术QCM-D来研究各种多肽和SBL的界面和运输问题，希望设计更有效的多肽并为抑制A？干扰奠定基础。PI计划测量两种不同家族的两亲性多肽(抗微生物、AMPs和淀粉样蛋白A？)和模型膜(SLB)之间的界面相互作用和运输，以发现更有效的AMP，并为防止A？破坏奠定基础。他们的实验攻击计划、材料选择和组合测量技术都是新颖的。还没有人制定出被广泛接受的多肽诱导像SLB这样的模型细胞膜破裂的机制的规则。我们的新方法将测量两个独立的参数，分别是频率和耗散的变化，或质量和刚性的变化，以及结合动力学。他们最近取得了进展，并首次观察到使用AMP(Picidin 1和Amp；3)和A？纤丝对SLB的破坏。在低浓度时，Picidin 1和Amp；3似乎形成孔状结构，而在高浓度时，它们提取部分双层并在SLB中形成胶束和大空腔。拟议研究的结果很重要，因为它们将有助于建立更有效地破坏细菌膜完整性的设计原则，并制定保护哺乳动物细胞膜免受多肽或低聚物破坏的策略。两个具体目标是：目的I：研究AMPS对类细菌SLB的破坏机制；目的II：研究A？1-42齐聚物物种对类哺乳动物SLM的崩解机制。这项拟议的研究将为多肽与SLB的相互作用提供一个基本的理解。这与灭活细菌感染和减轻淀粉样蛋白疾病具有广泛的相关性，如阿尔茨海默氏症、S、亨廷顿、S、帕金森、S和其他20多人。随着细菌对抗生素的耐药性增加，迫切需要新的和不同的方法来应对细菌感染。用AMPS灭活细菌提供了一种令人兴奋的抗生素替代品。这项研究的结果将通过优化AMP的治疗设计和涂覆医院墙壁来灭活空气中的细菌而造福社会。该项目将通过Rensselaer本科研究计划让本科理工科专业的学生参与，并通过RPI Questar计划让高中高年级学生参与，从而促进培训和学习。将再次招募女性和少数族裔学生，以扩大代表不足群体的参与，让学生接触现代界面科学。

This EAGER project is "high risk high payoff" in that it involves a radically new approach to investigate the mechanism of peptide-lipid interfacial interactions using a freely oscillating sensor called a Quartz Crystal Microbalance with Dissipation (QCM-D). Life depends on the integrity of biological membranes and their ability to maintain function. Intellectual Merit. Disrupting bacterial membrane integrity with peptides on purpose is a new strategy to kill toxic bacteria. Alternately, protecting mammalian cell membrane from disruption by peptides or oligomers is a strategy to mitigate Alzheimer?s (and many other amyloid-related) disease(s) (AD). In both cases, very little is known about how the peptides bind to biological membranes (or to membrane mimics called supported lipid bilayers, SLBs) and how they aggregate and penetrate to form pores and/or induce lipid loss. Various models of interaction include the formation peptide adsorbed on a SLB surface, pore formation with and without release of lipids from a SLB. Previously, methods such as interfacial tension (Langmuir-Blodgett (LB)), leakage, spectroscopy (NMR, EPR, CD) and scattering measurements (reflectometry with light, X-rays and neutrons) have been used to analyze protein-lipid interactions. Limitations include the presence of a nonnatural lipid-air interface (LB) and low sensitivity (NMR, EPR). Leakage measurements using fluorescence or electrical current changes are useful for determining the contiguity of the SBLs but do not provide much mechanistic insight. Scattering techniques are complementary to the proposed QCM-D measurements and will be pursued at Oak Ridge National Labs (our proposal to conduct neutron reflectometry in the Fall 2012 was successful) on the same problem described here. This is a complex interfacial binding, disruption and transport challenge, which occurs at the surface of SLBs. The PIs propose the use of a novel technique, QCM-D, to interrogate this interfacial and transport problem with a variety of peptides and SBLs with the hope of designing more potent peptides and establishing a foundation for inhibiting Aâ disruption.The PIs plan to measure the interfacial interactions and transport between two different families of amphipathic peptides (anti-microbial, AMPs, and amyloid protein, Aâ) and model membranes (SLBs) in order to discover more potent AMPs and establish a basis to prevent Aâ disruption.Their experimental plan of attack, materials choice and technique of measurement in combination are novel. No one has developed rules for a widely accepted mechanism of peptideinduced disruption of model cell membranes like SLBs. Our new approach will measure two independent parameters, changes in frequency and dissipation, or changes in mass and rigidity, respectively, and binding kinetics. They have recently made progress and observed disruption of SLBs with both AMPs (picidin 1 & 3) and Aâ fibrils for the first time. At low concentrations, picidin 1 & 3 appear to form porelike structures while at high concentrations, they extract parts of the bilayer and form micelles and large cavities in the SLB. The results from the proposed study are important because they will help establish design principles with greater efficacy to disrupt bacterial membrane integrity and formulate strategies to protect mammalian cell membranes from disruption by peptides or oligomers. The two specific goals are: Aim I: Investigate the rupture mechanism of bacterial-like SLBs by AMPS, and Aim II: Investigate the disintegration mechanism of mammalian-like SLMs by Aâ1-42 oligomer species.Broader Impact. The proposed study will provide a fundamental understanding of the interactions of peptides with SLBs. This has broad relevance to inactivating bacterial infections and to mitigating amyloid diseases like Alzheimer?s, Huntington?s, Parkinson?s and over 20 others. As bacteria increase resistance to antibiotics, new and different methods are urgently needed to deal with bacterial infection. Deactivating bacteria with AMPs offers an exciting alterative to antibiotics. Results of this research will benefit society by optimizing AMP design for treatment and for coating hospital walls to deactivate airborn bacteria. The project will promote training and learning by involving undergraduate science and engineering majors through the Rensselaer Undergraduate Research Program and by involving high school seniors through the RPI Questar program. Female and minority students will again be recruited to broaden participation of underrepresented groups, exposing students to modern interfacial science.