Structure-function studies of antimicrobial and fusogenic peptides by solid state NMR spectroscopy and MD simulation

背景：从生物学中学习，这项工作的重点是设计多功能和新型抗生素，以天然抗菌活性多肽为基础，具有智能设计和交付的巨大潜力--这将解决即使在发达国家也是抗击感染的一个主要卫生保健问题。一些细菌感染是新发现的，可用来控制它们的手段有限(MRSA，C.diff，S.Bureau)，特别是在老年人和免疫系统受损的人中，而其他更成熟的细菌感染由于(在一些国家)过多地开出已知抗生素的处方而产生耐药性。已知一些高效的天然抗菌肽(AMPs)，特别是从两栖类表皮(蛙皮)中提取的抗菌肽，了解它们的作用机制对设计新的AMPs有重要帮助。除此之外，天然蛋白质(特别是来自病毒的蛋白质)能够穿透细胞的外膜，有效地将其货物运送到新的宿主中。在这些通过进化而高度发展的系统的基础上，我们将使用自下而上的方法来设计新的AMP，使用天然(21种可用)和非天然(无限多样性)氨基酸。固体核磁共振(牛津)将被用来给出高分辨率(亚拉)距离限制，并帮助定义肽的二级结构(螺旋，β-折叠)，关于折叠和稳定性的信息，肽与脂类的分子特异性相互作用的细节，以及膜扰动。分子动力学(爱丁堡)将帮助进行初始的多肽设计，然后对输入的实验数据进行合理化，这些数据还将来自FTIR和CD研究(NPL)以及来自电子显微镜的样品形态。各种实验方法的样本优化将介于牛津和NPL之间，使用这两个实验室积累的大量经验。因此，这项建议将三个具有高度互补性的专业知识的成熟研究团队聚集在一起，专注于基础和分子科学水平的主要医疗保健问题。目的和目标：最初选择的系统是已知的AMP，具有膜结合和破坏的基本元素，即Maganin家族的多肽。序列信息将被用来设计新的Maganins同源物，但通过合理插入或改变氨基酸来改变功能。与此相关的是对来自HIV-1病毒的一种小蛋白gp41的研究，这种小蛋白具有膜活性特性，即膜扰动，因此具有穿透和/或吸收细胞的潜力。最终目标是从根本上了解新的潜在抗生素所需的设计原则，这些设计原则可以一直跟踪到临床试验和市场。潜在的应用和好处：这种NPL/EPSRC应用有两个关键组成部分，首先使NPL获得新的最先进的高分辨率(子)距离测量方法，使用世界上最高的场和专门的固态核磁共振仪器之一(牛津大学)，第二，加入一个新的NPL牵头的国际财团，致力于“生物物理系统中的长度-尺度桥接测量”，具有强大的未来商机和尖端研究。潜在的应用是通过生产新设计的AMP，这种AMP可以很好地用于对抗细菌耐药性，并提供了通过灵活的设计或通过避免耐药性的通用特性来解决和克服耐药性的原则。这项研究的好处显然是多种多样的，从对各种情况下膜-蛋白质相互作用的学术兴趣，到对患者的治疗用途。显然，如果我们要了解机制并设计新的方法来对抗微生物耐药性，就需要新的途径和智力投入，而这里建议的方法提供了这样的机会，具有潜在的明显好处。

Context: Learning from biology, the focus for this work is the design of versatile and novel antibiotics, based around natural antimicrobial-active peptides, with significant potential for intelligent design and delivery - this will address a major health-care problem, even in developed countries, of fighting infection. Some bacterial infections are newly discovered with limited means available to control them (MRSA, C. diff, S. bureau), especially in the aged and in those with compromised immune systems, and other more established bacterial infections have developed resistance due to over (self in some countries) prescription of known antibiotics. Some highly effective natural antimicrobial peptides (AMPs) are known, notably from amphibian epidermis (frog skin), and understanding the mechanism of their action can help significantly in the design of new AMPs. Added to this, natural proteins (notably from viruses) are capable of penetrating the outer membrane of cells, effectively delivering their cargo into a new host. Building on these highly developed systems through evolution, we will use a bottom-up approach to design new AMPs using both natural (21 are available) and unnatural (unlimited diversity) amino acids.Solid state NMR (Oxford) will be used to give high resolution (sub-Å) distance constraints and help define peptide secondary structure (helices, beta-sheets), information about folding and stability, details of molecularly specific interactions of peptides with lipids, and membrane perturbation. Molecular dynamics (Edinburgh) will aid in initial peptide design, and then rationalization of input experimental data which will also come FTIR and CD studies (NPL) and sample morphology coming from TEM. Sample optimization for various experimental methods will be between Oxford and NPL, using significant cumulative experience from both labs. This proposal therefore brings together three well-established research teams with highly complementary expertise to focus on a major health-care problem at the fundamental and molecular sciences level.Aims and Objectives: The systems of choice, initially, are known AMPs with essential elements of membrane association and disruption, namely peptides in the maganin family. Sequence information will be used to design new homologues of maganins, but with rationally inserted or changed amino acids to change function. Coupled to this will be studies of a small protein, gp41, derived from the HIV-1 virus with membrane active properties, namely membrane perturbing and hence potential for cell penetration and/or uptake.The final goal is to gain a fundamental understanding of the design principles required for new potential antibiotics which can be followed through to clinical trials and market.Potential applications and benefits: This NPL/EPSRC application has two key components, firstly bringing to NPL access to new state-of-the-art high resolution (sub-Å) distance measurement methodology, with one of the world's highest field and specialized solid state NMR instruments (at Oxford), and secondly joining a new NPL lead international consortium on "Length-scale Bridging Measurements in Biophysical Systems", with strong future business opportunities and cutting-edge research. The potential applications are through the production of newly designed AMPs which could fine use in combating bacterial resistance, and give principles on which resistance can be addressed and overcome, either through flexible design or through generalized properties which avoid resistance. The benefits of the research are clearly varied, from academic interest of membrane-protein interactions in all its multitude of situations, through to therapeutic use for the patient. Clearly new avenues and intellectual input is required if we are to understand the mechanisms and devise new ways to combat microbial resistance, and the approaches suggested here offer such opportunities, with potential obvious benefit.