Interactions in complex biological systems by nuclear magnetic resonance

细胞壁是药物和污染物遇到的第一道屏障，因此，生物活性分子可以靶向或穿越细胞壁，改变其结构，对细胞产生严重影响。同样，细胞器也被膜分隔开，在那里发生重要的毒性机制。因此，了解抗生素如何与细菌相互作用，或污染物如何进入微藻，将有助于解决细菌耐药性或环境问题。因此，掌握与细胞成分相互作用的纳米级原位信息是至关重要的。因此，我们研究计划的总体目标是开发新的固态核磁共振（SS-NMR）方法来表征完整细胞并研究它们在体内的分子相互作用。细胞内SS-NMR具有挑战性，因为大量细胞成分会产生大量信号。因此，我们研究计划的短期目标是建立在复杂细胞系统中实现光谱选择性和改善信号分配的策略，并开发监测分子与细胞相互作用的方法。为此，我们将重点关注人类和海洋细菌以及微藻。将使用两种方法：(1)对特定细胞成分进行排他性同位素标记；(2)结合核磁共振信号滤波的非特异性细胞标记。为了便于峰分配，将分离细胞成分（如细胞壁、叶绿体或线粒体）。这种重建方法将有助于整个细胞的数据收集和分析。该计划的长期目标是将我们的SS-NMR策略扩展到与高等生物分子相互作用的研究。“全细胞”核磁共振是迈向“整个生物体”核磁共振的垫脚石。我们的研究项目是创新的，因为它将把细胞内核磁共振的新兴领域扩展到世界上很少有研究小组做出贡献的细胞相互作用的研究。建立新方法的努力有可能将应用扩大到几个复杂的生物系统。这是值得的，因为核磁共振可以以非破坏性的方式告知结构和相互作用。我们的长期计划开辟了覆盖几种人类和动物病原体，陆地和水生微生物，甚至动物细胞系的可能性。这将有助于为设计新的抗生素提供一个新的工具箱，例如我们将研究的抗菌肽。由于全球气候变暖，具有多重耐药性的水生病原体正日益威胁着人类和水生生物群。通过研究水生细菌，我们的项目将帮助支持水产养殖业，这是加拿大沿海地区经济的重要部门。它还将使微藻细胞的密切表征，从而有助于其生物技术开发。

The cell wall is the first barrier encountered by drugs and contaminants and, as such, is targeted or traversed by bioactive molecules that can alter its architecture with critical consequences on the cell. Likewise organelles are delimitated by membranes where important toxicity mechanisms take place. Therefore understanding, for example, how antibiotics interact with bacteria, or pollutants enter microalgae, will help address bacterial drug resistance or environmental issues. It is thus crucial to have in situ nanometer-level information on interactions occurring with the cell constituents. The general objective of our research program is thus to develop new solid-state nuclear magnetic resonance (SS-NMR) approaches to characterize intact cells and study their molecular interactions in vivo. In-cell SS-NMR is challenging because of the tremendous number of signals that are produced by a huge number of cell constituents. Therefore the short-term objectives of our research program are to establish strategies to achieve spectral selectivity and improve signal assignment in complex cell systems, and to develop methodologies to monitor molecular interactions with cells. To do so, we will focus on human and marine bacteria as well as microalgae. Two approaches will be used: (1) exclusive isotopic labelling of specific cell constituents; and (2) unspecific cell labelling combined to NMR signal filtering. To facilitate peak assignment, cell constituents (such as cell wall, chloroplasts or mitochondria) will be isolated. This reconstructive approach will facilitate data collection and analysis in whole cells. The long term objective of this program is to expand our SS-NMR strategy to the study of molecular interactions with higher organisms. “Whole-cell” NMR is the stepping stone towards “whole-organism” NMR. Our research program is innovative because it will extend the emerging field of in-cell NMR to the study of cell interactions to which very few research groups in the world have contributed. Efforts in establishing new methodologies have the potential to expand applications to several complex biological systems. This is worthwhile since NMR can inform on structure and interactions in a non-destructive manner. Our long-term program opens the possibility to cover several human and animal pathogens, terrestrial and aquatic microorganisms, and even animal cell lines. It will help provide a novel toolbox for the design of new antibiotics, such as antimicrobial peptides that we will study. Because of global warming, aquatic pathogens subject to multi-drug resistance are increasingly threatening humans and the water biota. By studying aquatic bacteria, our program will help support aquaculture an important sector for the economy of Canadian coastal regions. It will also enable close characterization of microalgal cells, thus helping their biotechnological exploitation.