SFG Investigation of Peptide and Protein Orientations in Membranes

描述(申请人提供)：这项研究将开发一种系统的方法，使用联合和频率产生(SFG)、双共振SFG(DRSFG)、衰减全反射FTIR(ATR-FTIR)和四波混频(FWM)研究α-螺旋结构，以原位表征单一脂质双层中的膜肽/蛋白质取向。该方法可以推断膜肽或蛋白质在接近真实膜环境的单一脂双层中的绝对取向和复杂取向分布。它还可以研究脂双层的不对称性对多肽取向的影响，并可以表征表面覆盖率很小的多肽的结构信息。本研究获得的各种抗菌肽在脂质双层中的定位有助于阐明这些抗菌肽在膜上的作用方式。抗生素耐药性是目前医学上最紧迫的问题之一，我们相信这项研究将对抗菌肽的设计和优化产生影响。该方法还将应用于研究G蛋白亚单位在不同环境中的取向，为了解受体和G蛋白在信号转导过程中如何在膜上组织提供独特的见解，并为各种疾病(如心力衰竭)提供基本见解。其具体目的是：1.SFG研究辅以ATR-FTIR和FWM研究，可以提供单一脂双层中不同膜肽的唯一定位信息。这些研究将导致膜环境中更详细的多肽取向分布的确定。这一结果也将为在特定目标2中提出的研究提供校准基础。2.将DRSFG用于特定目标1中检测的多肽的研究，以表明DRSFG可以极大地提高正常SFG的灵敏度。利用DRSFG可以表征表面浓度极低的膜肽的独特结构信息(肽脂摩尔比1：5,000)。3.除了-螺旋多肽外，还将研究蛋白质中的-螺旋结构，以论证SFG结合ATR-FTIR和FWM确定膜蛋白二级结构域结构信息和膜蛋白取向的可行性。本研究将以三聚体G蛋白的G？1？2亚基为模型。公共卫生相关性在这项研究中，振动光谱技术的组合可以提供关于膜多肽和蛋白质的重要定位信息，这是以其他方式很难获得的。这些工作有助于深入了解抗菌肽和G蛋白的膜取向，为开发治疗传染病、心脏病、哮喘、阿片成瘾和高血压的药物提供重要信息。

DESCRIPTION (provided by applicant): This research will develop a systematic way to use combined sum frequency generation (SFG), double resonance SFG (DRSFG), attenuated total refection FTIR (ATR-FTIR), and four-wave mixing (FWM) studies on alpha-helical structures to characterize membrane peptide/protein orientation in a single lipid bilayer in situ. The methodology can deduce the absolute orientation and complicated orientation distribution for a membrane peptide or protein in a single lipid bilayer that closely resembles the real membrane environment. It can also study the effect of asymmetry in lipid bilayers on peptide orientations, and can characterize structural information of peptides with very small surface coverage. The orientations of various antimicrobial peptides in lipid bilayers obtained in this study can help to elucidate modes of actions of such peptides on membranes. Antibiotic resistance is one of the most pressing problems in medicine at present, and we believe that this research will impact the design and optimization of peptides for antimicrobial purposes. The methodology will also be applied to study orientations of subunits of G-proteins in various environments, lending unique insight into how receptors and G proteins are organized in membranes during signal transduction and providing fundamental insights into various diseases such as cardiac failure. The specific aims are: 1. SFG studies supplemented by ATR-FTIR and FWM research can provide unique orientational information of various membrane peptides in a single lipid bilayer. These studies will lead to the determination of more detailed orientation distribution of the peptides in the membrane environment. The result here will also provide a calibration base for the studies proposed in Specific Aim 2. 2. DRSFG will be used to investigate the peptides examined in Specific Aim 1 to show that DRSFG can greatly improve the sensitivity of normal SFG. Unique structural information of membrane peptides with a very low surface concentration (peptide-lipid molar ratio<1:5,000) can be characterized using DRSFG. 3. In addition to ?-helical peptides, ?-helical structures in proteins will also be investigated to demonstrate the feasibility of determining structural information of secondary structural domains of membrane proteins and the orientation of membrane proteins using SFG, supplemented by ATR-FTIR and FWM. The G?1?2 subunit of a trimeric G-protein will be used as a model in this research. PUBLIC HEALTH RELEVANCE In this research a combination of vibrational spectroscopic techniques can provide vital orientational information regarding membrane peptides and proteins, which is difficult to obtain otherwise. Such work enables in-depth understanding of membrane orientations of antimicrobial peptides and G-proteins, providing important information to develop cures for infectious diseases, heart disease, asthma, opioid addiction, and hypertension.