Protein Biophysics in Cells

细胞中的蛋白质生物物理学

• 批准号: 0212939
• 负责人: Gary Pielak
• 金额: $ 44.67万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212939&HistoricalAwards=false
• 关键词: Protein; Biophysics; Cells

The inside of a cell is a crowded place where macromolecular solutes, mostly proteins, reach concentrations of hundreds of grams per liter. However, almost all in vitro studies of protein biophysics are conducted at solute concentrations that rarely exceed a fraction of a gram per liter. This discrepancy poses an important question: do the observations made in dilute solution always coincide with what happens inside cells? Preliminary data show that the answer is no. Specifically, NMR data show that FlgM, a protein that is unfolded in dilute solution, gains structure inside living Escherichia coli cells. Additional data show that FlgM gains structure in vitro on adding high concentrations (400 g/L) of glucose, bovine serum albumin, or ovalbumin. Given these data proving the biological significance of crowding, the objective of the research is to understand the effects of physiologically relevant crowded environments on protein equilibria, particularly direct studies in living cells. The proteins to be studied are FlgM, alpha -synuclein, and cytochrome c. Like FlgM, alpha -synuclein is unfolded in dilute solution. The first hypothesis is that there are two classes of natively unfolded proteins. Class 1 (FlgM) collapses and folds under physiologically relevant crowded conditions. Class 2 (alpha -synuclein) collapses but does not fold under these conditions. The second hypothesis is that macromolecular crowding stabilizes globular proteins in cells. The globular protein cytochrome c will be used to test this hypothesis. The methods to be used include, heteronuclear multidimensional NMR, especially NMR detected amide proton exchange experiments, circular dichroism spectropolarimetry, and directed mutagenesis.

细胞内部是一个拥挤的地方，大分子溶质，主要是蛋白质，达到每升数百克的浓度。然而，几乎所有蛋白质生物物理学的体外研究都是在溶质浓度很少超过每升几分之一克的情况下进行的。这种差异提出了一个重要的问题：在稀溶液中进行的观察是否总是与细胞内发生的情况一致？具体来说，NMR数据显示FlgM是一种在稀溶液中展开的蛋白质，在活的大肠杆菌细胞内获得结构。另外的数据显示，FlgM在加入高浓度（400 g/L）的葡萄糖、牛血清白蛋白或卵清蛋白时在体外获得结构。鉴于这些数据证明了拥挤的生物学意义，研究的目的是了解生理相关的拥挤环境对蛋白质平衡的影响，特别是在活细胞中的直接研究。待研究的蛋白质是FlgM、α-突触核蛋白和细胞色素c。与FlgM一样，α-突触核蛋白在稀溶液中是未折叠的。第一个假设是存在两类天然未折叠蛋白质。类1（FlgM）的崩溃和折叠生理相关的拥挤条件下。2类（α-突触核蛋白）在这些条件下塌陷但不折叠。第二个假设是大分子拥挤稳定了细胞中的球状蛋白。球状蛋白质细胞色素c将用于检验这一假设。所使用的方法包括异相多维NMR，特别是NMR检测的酰胺质子交换实验、圆二色谱法和定向诱变。