Macromolecular crowding in vitro and in cells

细胞包含复杂而拥挤的大分子集合，其浓度可超过300克/升。因此，研究生物大分子最相关的环境是拥挤的环境。然而，我们对生物大分子的了解大多来自于大分子浓度低于10g /L的溶液。此外，许多理论预测拥挤会对生物大分子的性质产生很大的影响。这个项目将研究拥挤如何影响蛋白质的物理性质。将定量生物物理学从简单的解决方案转移到拥挤的环境，包括活细胞内部，是一个具有重要成果的重大挑战。这项工作将有助于培养本科生和研究生从事前沿研究的实践。此外，所获得的知识将增加对生物学的基本理解，并为生产设计酶和稳定基于蛋白质的试剂提供信息。这些努力是建立美国生物经济的关键。拟议研究的总体目标是了解活细胞和体外拥挤条件下蛋白质的分子生物物理学。首席研究员和他的实验室已经开发了基于核磁共振的工具来进行这些测量。要评估的蛋白质性质包括平衡、热力学稳定性和溶剂化。这项工作已经导致发现大分子之间的化学相互作用在拥挤中发挥的作用比以前认为的要大得多。首席研究员和他的学生同事现在将确定这些化学相互作用的来源，检查细胞内环境对无序蛋白质的影响，并通过研究多结构域蛋白酶，二氢叶酸还原酶，增强他们努力的生物学和生物技术相关性。

Cells contain a complex and crowded collection of macromolecules whose concentration can exceed 300 g/L. Thus, the most relevant environment for studying a biological macromolecule is a crowded one. Most of what is known about biological macromolecules, however, comes from solutions where the concentration of macromolecules is less than 10 g/L. Furthermore, many theories predict that crowding will have large affects on the properties of biological macromolecules. This project will investigate how crowding affects the physical properties of proteins. The goal of moving quantitative biophysics from simple solutions to crowded environments, including the inside of living cells, is a major challenge with important outcomes. The work will facilitate the training of undergraduate and graduate students in the practice of cutting-edge research. In addition, the knowledge gained will both add to the fundamental understanding of biology and inform efforts to produce designer enzymes and stabilize protein-based reagents. These efforts are key to building the US bioeconomy.The overarching objective of the proposed research is to understand the molecular biophysics of proteins in living cells and under crowded conditions in vitro. The principal investigator and his laboratory have developed nuclear magnetic resonance-based tools to make these measurements. The protein properties to be assessed include equilibrium thermodynamic stability and solvation. This work has already resulted in the discovery that chemical interactions between macromolecules play a much larger role in crowding than previously thought. The principal investigator and his student colleagues will now identify the source of these chemical interactions, examine the impact of the intracellular environment on disordered proteins and enhance the biological and biotechnological relevance of their efforts by studying the multidomain protein enzyme, dihydrofolate reductase.