Protein Biophysics in Cells

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，几乎所有的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：在稀溶液中观察到的结果与细胞内发生的事情总是一致的吗？初步数据显示，答案是否定的。具体地说，核磁共振数据显示，FlgM是一种在稀溶液中展开的蛋白质，它在活的大肠杆菌细胞中获得结构。更多的数据表明，在体外添加高浓度(400g/L)的葡萄糖、牛血清白蛋白或卵清蛋白后，FlgM获得了结构。鉴于这些数据证明了拥挤的生物学意义，这项研究的目的是了解与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。要研究的蛋白质是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展开。第一种假设是存在两类天然未折叠的蛋白质。第一类(FlgM)在生理上相关的拥挤条件下坍塌和折叠。在这些条件下，2类(α-突触核蛋白)折叠但不折叠。第二个假设是，大分子拥挤稳定了细胞中的球状蛋白。球状蛋白细胞色素c将用来检验这一假说。所使用的方法包括，异核多维核磁共振，特别是核磁共振检测的酰胺质子交换实验，圆二色谱偏振，定向诱变。

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.