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

内容：从生物学中学习，这项工作的重点是设计基于天然抗菌活性肽的多功能和新型抗生素，具有智能设计和交付的巨大潜力-这将解决一个主要的卫生保健问题，即使在发达国家，也是对抗感染。一些细菌感染是新发现的，可用于控制它们的手段有限（MRSA，C. diff，S.局），特别是在老年人和免疫系统受损的人中，以及其他更确定的细菌感染由于已知抗生素的过度（在一些国家中为自我）处方而产生了耐药性。已知一些高效的天然抗菌肽（AMP），特别是来自两栖动物表皮（青蛙皮肤），并且理解它们的作用机制可以显著帮助设计新的AMP。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，有效地将它们的货物运送到新的宿主中。在这些高度发达的系统的基础上，通过进化，我们将使用一种自下而上的方法来设计新的AMP，（21个可用）和非自然固态NMR（Oxford）将用于给出高分辨率（亚微米）距离约束并帮助定义肽二级结构（螺旋，β-折叠），折叠和稳定性的信息，肽与脂质的分子特异性相互作用的细节，和膜扰动。分子动力学（Edinburgh）将有助于初始肽设计，然后合理化输入实验数据，这些数据也将来自FTIR和CD研究（NPL）以及来自TEM的样品形态。各种实验方法的样品优化将在牛津和NPL之间进行，使用两个实验室的大量累积经验。因此，这项建议汇集了三个成熟的研究团队，具有高度互补的专业知识，专注于一个主要的医疗保健问题的基础和分子sciences level.Aims和Objectives：系统的选择，最初，是已知的AMP与膜协会和中断的基本要素，即在maganin家族的肽。序列信息将用于设计新的maganins同源物，但合理插入或改变氨基酸以改变功能。与此同时，研究人员还将对一种来自HIV-1病毒的小蛋白gp 41进行研究，该蛋白具有膜活性，即膜扰动，因此具有细胞渗透和/或吸收的潜力。最终目标是对新的潜在抗生素所需的设计原理有基本的了解，这些抗生素可以用于临床试验和市场。潜在的应用和好处：该NPL/EPSRC应用程序有两个关键组成部分，第一个是为NPL提供新的最先进的高分辨率（亚分辨率）距离测量方法，使用世界上最高的现场和专业固态NMR仪器之一（在牛津大学），其次加入了一个新的NPL牵头的国际财团“生物物理系统中的长度尺度桥接测量”，拥有强大的未来商业机会和尖端研究。潜在的应用是通过生产新设计的AMP，其可以很好地用于对抗细菌耐药性，并给出可以通过灵活的设计或通过避免耐药性的一般化性质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，从学术兴趣的膜蛋白质相互作用在其所有的众多情况下，通过对病人的治疗用途。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，这里建议的方法提供了这样的机会，具有潜在的明显益处。

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.