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

我们实验室基于NSERC的研究的主题是连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、分子水平、结构和动力学研究通常是在组成非常简单的系统中进行的，例如，一种类型的脂类和一种类型的蛋白质。然而，这些简单系统的结果与真实的、复杂得多的生物系统的结果相比，并不总是很清楚。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，并将它们扩展到更复杂的生物系统，以获得对现实生活中分子结构和动力学的重要见解。通过这次探索资助金的续签，我计划主要关注两个方面。目标1将建立在我们已经成功的NSERC关于抗菌肽(AMPs)如何与完整细菌相互作用的研究的基础上。AMPS是许多生物体先天免疫系统的重要组成部分，可以抵御包括细菌、病毒和微生物真核生物在内的各种入侵病原体。然而，我们对AMP如何工作的理解并没有超出它们与病原体的一种成分--脂质--相互作用的范围。在目标2中，我们将把我们在对复杂生物样本进行生物物理学方面的专业知识应用到我们的一个新领域，分子拥挤中的软相互作用，最初应用于固有无序蛋白质(IDPs)。分子拥挤，即细胞内生物分子浓度非常高的影响，是重要的，因为与通过传统生物化学实验在稀释条件下观察到的相比，它可以改变生物分子结构和功能。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；与用模型脂质系统观察脂质双层破坏所需的比率相比，AMP与脂质的比率约为10,000才能看到细胞生长的抑制。因此，找出AMP除了与脂类相互作用外，还与哪些成分相互作用，以及非脂相互作用如何改变AMP诱导的膜破坏是非常有意义的。我们在这一领域的早期工作引起了高度的兴趣，这一点从三个关于这个主题的受邀审查中得到了证明，我们预计这种高度的兴趣将继续下去。我们的目标2的结果将通过建立一个迫切需要的新桥梁来影响该领域，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真实细胞。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，我们的工作将提供对通过细胞内核磁共振获得任何类型蛋白质的高质量数据感兴趣的参数的宝贵见解。

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.