Bridging molecular studies of model and real biological systems with biophysics experiments on complex samples

我们实验室基于nserc的研究主题是桥接模型和真实的生物系统。这种方法的基本原理是，对生物分子的详细的、分子水平的、结构和动力学研究通常是用非常简单的组成系统来完成的，例如，用一种脂质和一种蛋白质。然而，人们并不总是清楚这些简单系统的结果如何与真实的、更复杂的生物系统中的结果相比较。因此，我们的方法是在为简单系统开发的生物物理方法之间建立桥梁，并将其扩展到更复杂的生物系统，以便获得对现实生活中的分子结构和动力学的重要见解。随着探索基金的续期，我计划重点关注两个方面。目标1将建立在我们已经成功的NSERC研究抗菌肽（amp）如何与完整细菌相互作用的基础上。amp是许多生物体先天免疫系统的重要组成部分，可以抵御各种入侵病原体，包括细菌，病毒和微生物真核生物。然而，我们对amp如何工作的理解并没有延伸到它们如何与病原体的一种成分：脂质相互作用。在目标2中，我们将把我们在复杂生物样品中进行生物物理学的专业知识应用到我们的新领域，分子拥挤中的软相互作用，最初应用于内在无序蛋白（IDPs）。分子拥挤，即细胞内高浓度生物分子的影响，是很重要的，因为与传统生物化学实验在稀释条件下观察到的情况相比，它可以改变生物分子的结构和功能。目标1的潜在影响涉及用于研究AMP分子机制的条件与实际观察生物功能的实验之间的巨大差异；与在模型脂质系统中观察脂质双分子层破坏所需的比率相比，AMP与脂质之比要大10000才能观察到细胞生长的抑制。因此，除了脂质外，AMP还与哪些成分相互作用，以及非脂质相互作用如何修饰AMP诱导的膜破坏是非常有趣的。我们在这一领域工作的早期阶段受到了高度关注，关于该主题的三次受邀评论证明了这一点，我们希望这种高度关注能继续下去。我们的目标2的结果将通过建立一个急需的和新颖的桥梁来影响该领域，以更好地理解如何将在简单拥挤系统上进行的大量工作应用于实际细胞。相反，我们的结果也应该有助于解释从细胞内实验中收集到的观察结果。此外，我们的工作将为通过细胞内核磁共振获得任何种类蛋白质的高质量数据提供有价值的见解。

The theme of the NSERC-based research in our lab is bridging model and real biological systems. The rationale for this approach is that detailed, molecular level, structural and dynamics research on biological molecules is generally done with systems of very simple composition, e.g. with one type of lipid and one type of protein. However, it's not always clear how the results from these simple systems compare to what goes on in the real, much more complex biological systems. Thus, our approach is to build bridges between the biophysical methods that have been developed for simple systems and extend them to much more complex biological systems in order to gain important insights into real life molecular structure and dynamics. With this Discovery Grant renewal, I am planning two main foci. Objective 1 will build on our already successful NSERC research into how antimicrobial peptides (AMPs) interact with intact bacteria. AMPs are a crucial component of the innate immune system of many organisms and can protect against a variety of invading pathogens including bacteria, viruses, and microbial eukaryotes. However, our understanding of how AMPs work does not extend much past how they interact with one component of pathogens: the lipids. With Objective 2, we will apply our expertise in performing biophysics with complex biological samples into a new area to us, soft interactions in molecular crowding, initially as applied to Intrinsically Disordered Proteins (IDPs). Molecular crowding, i.e. the effect of the very high concentration of biomolecules inside cells, is important because it can modify the structure and function of biological molecules compared to what is observed in dilute conditions via traditional biochemistry experiments.The potential impact of Objective 1 relates to the enormous disparity between conditions used for studies of AMP molecular mechanisms, versus experiments where biological functional is actually observed; it takes an AMP to lipid ratio of ~10,000 greater to see inhibition of cell growth as compared to the ratio needed to see lipid bilayer disruption with a model lipid system. Thus, it is of high interest to find out with which components, besides the lipids, the AMP is interacting and how the non-lipid interactions modify the AMP-induced membrane disruption. There has been a high level of interest in the earlier phase of our work in this area, as evidenced by three invited reviews on the topic, and we expect this high interest to continue.Our results from Objective 2 will impact the field via building a much needed and novel bridge to better understand how the large body of work performed on simple crowding systems can be applied to real cells. Conversely, our results should also help explain observations gleaned from in-cell experiments. Additionally, our work will provide valuable insights into parameters of interest in getting quality data with any kind of protein via in-cell NMR.